WO1986005518A1 - Stereoregular polynucleotide-binding polymers - Google Patents

Stereoregular polynucleotide-binding polymers Download PDF

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WO1986005518A1
WO1986005518A1 PCT/US1986/000544 US8600544W WO8605518A1 WO 1986005518 A1 WO1986005518 A1 WO 1986005518A1 US 8600544 W US8600544 W US 8600544W WO 8605518 A1 WO8605518 A1 WO 8605518A1
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backbone
sequence
polymer
moieties
target
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PCT/US1986/000544
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French (fr)
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James Summerton
Dwight Weller
Eugene Stirchak
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James Summerton
Dwight Weller
Eugene Stirchak
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Priority to AT86902189T priority Critical patent/ATE81872T1/en
Priority to DE8686902189T priority patent/DE3687030T2/en
Publication of WO1986005518A1 publication Critical patent/WO1986005518A1/en

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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to base-specific polynucleotide-binding polymers
  • Compounds capable of recognizing and binding specifically to a target base sequence in a polynucleotide have many potentially valuable applications.
  • the compounds could act to bind to and inactivate viral genomes, or other single-stranded polynucleotide species characteristic of an infecting microorganism.
  • the compounds could be used to "hybridize" with analyte single-stranded polynucleotides, to detect the presence of viral or bacterial-specific nucleic acid species.
  • recombinant DMA methods have allowed for the design of plasmids which carry a anti-sense gene whose RNA transcript can recognize and bind to a particular target single-strand polynucleotide.
  • Introduction of such a plasmid into cells results in continuing production of anti-sense mRNA which appears to pair with its complementary target mRNA and thereby block its translation (Izant and Weintraub (1984), Cell, 36:1007; Izant and Weintraub (1985), Science, 229 :345).
  • a particular merit of this approach is that successful introduction of even a single such plasmid could potentially effect continued inactivation of the targeted gene activity and/or permanent resistance to the pathogen containing the targeted genetic sequence.
  • the approach is severely limited, however, by the problems of transforming a large number of tissue or organ cells with the plasmid construct.
  • uncharged and substantially uncharged heteropolymeric polynucleotide analogs more nearly isostructural with nucleic acids have been prepared for the purpose of in vivo inactivation of targeted single-stranded genetic sequences.
  • These substances include both aryl nitrogen mustard derivatized (Karpova et al (1980), FEBS Letters, 122:21) and underivatized (Richard Tullis, Personal. Communication, Molecular Biosystems, Inc., San Diego) oligonucleotides having their phosphates in the uncharged triester state.
  • a novel class of polynucleotide analogs which has recently been reported contain predominantly uncharged, atactic methylephosphonate backbone linkages, or homochiral methylphosphonate linkages alternating with charged, achiral phosphodiester linkages (Miller, Yano, Yano, Caroll, Jayaraman, and Ts'o (1979), Biochem., 18:5134; Miller, Dreon, Pulford, and Parland (1980), J. Biol.
  • oligonucleotide analogs have been reported to enter living cells and pair with complementary genetic sequences therein.
  • optimal uptake by the cells is achieved if the compounds are designed with one or a few ionic groups per molecule, to enhance solubility in aqueous medium. If the charge density on the backbone is increased significantly, however, problems of poor transport across the cell membrane are encountered. Also, greater stability to nucleases is achieved if phosphodiester linkages are avoided.
  • the above uncharged and substantially uncharged polynucleotide analogs all have chiral backbone linkages, i.e., linkages in which two possible stereoisomeric forms are allowed.
  • the backbone of the polymer produced by linking subunits has a random sequence of left- and right- handed forms.
  • This type of backbone structure which is also referred to as an atactic backbone, is characterized by a broad range of analog/target binding constants.
  • the mean binding constant can be significantly reduced with respect to the binding constant between normal complementary polynucleotides.
  • oligonucleotide analogs having tactic linkages could be prepared by means of a geometric assembly procedure in which one of the two stereoisomers formed at each subunit addition step is retained, and the other discarded.
  • the limitation of this procedure is that at least half of the material is discarded at each subunit addition step, so that the yield can be at most 1/2 of the starting material, where n is the number of rounds of couplings required in the synthesis.
  • composition which can be designed for targeting against virtually any nucleotide target sequence within a length range of between about 5-30 base pairs, and which can be prepared in relatively high yield;
  • the invention includes a polymeric composition designed to bind, with a selected binding affinity, to a single-stranded polynucleotide containing a target sequence of bases.
  • the composition includes non-homopolymeric, substantially stereoregular polymer molecules of the form:
  • R I -R n are recognition moieties selected from purine, purine-like, pyrimidine, and pyrimidine like heterocycles effective to bind by Watson/Crick pairing to corresponding, in-sequence bases in the target sequence;
  • n is such that the total number of Watson/Crick hydrogen bonds formed between a polymer molecule and target sequence is at least about 15;
  • B ⁇ 8 are backbone moieties joined by chemically stable, substantially uncharged, predominantly achiral linkages
  • the unit length of backbone moieties is 5-7 atoms if the backbone moieties have a cyclic structure, and 4-6 atoms if the backbone moieties have an acyclic structure;
  • the backbone moieties support the recognition moieties at positions which allow Watson/Crick base pairing between the recognition moieties and the corresponding, in-sequence bases of the target sequences.
  • the polymeric molecules have a small number of charges, to increase compound solubility in aqueous medium.
  • the charges may be attached at end regions of the molecules or at achiral charged backbone linkages.
  • the polymer molecules are constructed, according to the method of synthesis of the invention, by first selecting a sequence of bases in the target polynucleotide.
  • the sequence is typically between about 5-32 bases, and preferably between about 8-20 bases, corresponding to the number of subunits in the final binding polymer.
  • the binding affinity of the binding polymer for the target polynucleotide can be increased by increasing the number of target-sequence bases (and correspondingly, the number of subunits in the binding polymer).
  • Polymer subunits of the form B-R i and/or B-R j are then coupled in a defined sequence corresponding to the sequence of bases in the target-specific sequence.
  • R i and R j are recognition moities, as defined above, which are capable of forming Watson/Crick base pairing with corresponding polynucleotide bases through either two or three hydrogen bonds, respectively.
  • the binding affinity or the Tm of the binding polymer for the target polynucleotide can be selectively varied according to the ratio of R i to R j recognition moieties used in forming the polymer, with a greater percentage of R i recognition moieties producing a lower binding affinity for the target polynucleotide.
  • the polymers are useful in a diagnostic system for detecting an analyte polynucleotide having a defined target base sequence.
  • the system includes a support reagent composed of the polymer molecules of the invention linked to a solid support.
  • the polymer binding molecules are predominantly uncharged.
  • the backbone moieties are joined by achiral charged linkages, such as phosphodiester linkages, alternating with one or more uncharged achiral linkages.
  • a polynucleotide analyte is added to the diagnostic reagent under conditions in which the analyte is in a single-strand form and which allow sequence-specific pairing between the analyte and the reagent polymers.
  • the reagent is washed to remove unbound test material.
  • the reagent and bound analyte are then reacted with a oligocationic reporter which is designed to bind by charge attraction to the charged, phosphodiester linkages of the bound analyte, but not to the uncharged or substantially uncharged polymer molecules.
  • the ratio of bound reporter to bound analyte polynucleotides can be 10 2 -10 4 , giving a highly sensitive assay.
  • the polymer composition is designed to inhibit expression of a selected target single-stranded polynucleotide, such as an RNA virus.
  • the polymer is preferably constructed to bind spsecifically to the target polynucleotide, with the minimum binding needed to produce the desired inhibition of target expression, to minimize non-specific binding effects.
  • Figure 1 shows preferred purine and pyrimidine structures used in forming polymer molecules of the invention
  • Figure 2 shows preferred purine-like and pyrimidine-like recognition moieties used in forming the polymer molecules
  • Figure 3 shows preferred cyclic backbone moities used in forming the polymer molecules
  • Figure 4 shows preferred acyclic backbone moieties used in forming the polymer molecules
  • Figures 5A and 5B illustrate two preferred subunit assembly schemes for coupling N 1 ---N 2 type subunit backbones
  • Figure 6 shows the backbone structures of subunit dimers A-A through D-D formed in accordance with the methods illustrated in Figures 5A and 5B, and two cyclic backbone structures formed in accordance with the methods illustrated in Figures 7A and 7B;
  • Figures 7A-7C illustrate three preferred subunit assembly schemes for coupling N 1 ---E type subunit backbones.
  • Figure 8 shows preferred acyclic backbone structures of subunit dimers formed in accordance with the methods illustrated in Figures 7A-7C;
  • the polymeric composition of the invention is designed to bind, with a selected binding affinity, to a polynucleotide containing a target sequence of bases.
  • the composition is composed of non-homopolymeric, substantially stereoregular molecules or species of the form:
  • B-R i are a series of base-specific subunits containing a backbone moiety B and a recognition moiety
  • R i which is selected to bind specifically by
  • Watson/Crick base pairing to a corresponding, in-sequence base in the target sequence, and the subunits are joined through their backbone moieties predominantly achiral, substantially uncharged bonds.
  • Section I Methods for coupling subunits through achiral linkages are described in Section II, with subunit-protective group strategies involved in subunit synthesis being discussed in Section II.
  • the polymer species are synthesized by sequential subunit coupling, to form polymer molecules of a selected length and sequence.
  • the length of the target sequence and polymer sequence is selected to achieve a desired binding specificity.
  • the binding affinity of the polymer for the target can be selectively varied by several strategies discussed in Section IV, including the choice of recognition moieties which are capable of forming either three (for greater binding affinity) or two (for lesser affinity) base-paired hydrogen bonds with the corresponding target base.
  • the resulting composition contains polymer species or molecules, all having substantially the same sequence of subunits and substantially the same sequence of intersubunit linkage types. In functional terms, all of the polymer species have substantially the same binding affinity for the target polynucleotide. Methods for assembling the polymer, once a desired subunit sequence is selected, are given in Section IV.
  • the polymer composition is useful in a novel solid-phase diagnostic system which is described in Section IV. This system is based on the binding of analyte polynucleotide molecules to support-bound polymer molecules and subsequent elctrostatic attachment of multiple polycationic reporter molecules to the polynucleotide, to give an amplified reporter signal for each bound analyte molecule.
  • the polymer is also useful in a variety of solution applications, including therapeutic applications, which are also considered in Section IV. I. Subunit Structure
  • the design of the subunits B-R i which are suitable for use in forming the polymer species of the invention involves a number of structural and/or stereochemical criteria which must be met both by the backbone and recognition moieties and by the linkage between the two. The design requirement of the recognition moiety will be considered first.
  • the recognition moiety of each subunit must provide two or three hydrogen-bonding groups held in an unambiguous configuration adapted for hydrogen bonding to two or three of the Watson/Crick hydrogen bonding sites on a specified in-sequence base of the target genetic sequence.
  • the tautomeric state of the recognition moiety should be relatively stable, and (2) the recognition moiety should have a structure which provides a relatively rigid arrangement of the hydrogen-bonding groups. Such rigidity is best afforded by a ring structure having the polar hydrogen-bonding groups either forming part of the ring, or directly attached to the ring.
  • the preferred recognition moiety structures include purine, purine-like, pyrimidine, and pyrimidine-like structures which are designed to form Watson/Crick base pairing, through either two or three hydrogen bonds, with selected polynucleotide bases.
  • the group of subunits used in polymer synthesis includes at least two recognition moieties which are base-specific for different polynucleotide bases, and preferably one or more recognition moieties for each of the four bases in a natural DNA or RNA polynucleotide.
  • FIG. 1 shows exemplary purine and pyrimidine type recognition moieties.
  • thymine or uracil bases structures 3-6, to guanine bases; structures 7-9, to cytosine bases, and structures 10-12, to adenine bases.
  • Structures 1, 4, 5, 6, 8, and 10-12 are R i type moieties adapted to bind to corresponding in-sequence bases through two hydrogen bonds, and the remaining structures are R j type moieties which form three hydrogen bonds on base pairing.
  • purine and pyrimidine nucleosides are useful in synthesizing a variety of other subunits which are suitable for use in polymer synthesis. These subunits, modified if necessary with amine protective groups, can be obtained from commercial sources, or prepared according to known methods, such as those described or referenced in Example 1.
  • Figure 2 These structures have base pairing specificities and hydrogen bonding properties like those of the analogous structures shown in Figure.
  • the binding properties of a polymer containing a carbon-linked recognition moiety may not be significantly different from that of a polymer whose recognition moities are nitrogen-linked, as in Figure 1, subunits incorporating the carbon-linked moieties generally require a greater synthetic effort than do subunits containing preformed purine and pyrimidines.
  • the carbon-linked recognition moieties provide a useful starting material.
  • each subunit has the general form N 1 ---E, or N 1 ---N 2 , where N 1 and
  • N 2 are nucleophilic groups and E is an electrophilic group.
  • the preferred nucleophilic groups are amino, hydroxyl and hydrazino; and the preferred electrophilic groups, and/or electrophilic linking agents, are derivatives of carbonic, thiocarbonic, carboxylic, and sulfonic acids.
  • Backbone moieties can have either a cyclic or an acyclic structure. While the total number of possible backbone moiety structures is very large, nevertheless, only a rather limited number of structures are of actual practical value due to a number of factors. The initial procedure by which promising backbone moieties were selected was as follows.
  • Each resulting polynucleotide/polymer duplex was then examined for coplanarity of the Watson/Crick base-pairs, torsional and angle strain in the prospective polymer backbone, degree of distortion imposed on the nucleic acid strand, and interstrand and intrastrand nonbonded interactions. Special attention was paid to whether or not each amide-containing backbone could readily adopt a conformation wherein its amide moieties were planar. This is important because of the substantial energy cost required to force an amide into a nonplanar conformation.
  • the required unit backbone length i.e., the N 1 ---E spacing in an N 1 ---E or activated N 1 ---N 2 -E subunit
  • the required unit backbone length is 5-7 atoms for backbone moieties constituting or derived from deoxyribose or ribose (cyclic backbone structures), with a 6-atom length being optimal.
  • Subunit structure judged acceptable in the above modeling studies were then assessed for feasibility of synthesis (on the basis of key synthetic reactions reported in the literature, or reactions carried out on model compounds, and/or via actual synthesis of the subunits) and stability of the assembled polymer backbone (preliminary stability studies were generally carried out with suitably linked model compounds or subunit dimers). These types of studies were used to further restrict the number of candidate backbone structures. From this restricted candidate pool the cyclic backbone structures A-G shown in Figure 3 were, ultimately selected as preferred structures.
  • subunits A-D containing N 1 ----N 2 type cyclic backbone moieties include:
  • N 1 ---E-type backbone moieties include
  • acylic backbone moiety structures I-N shown in Figure 4 all of which contain a one-atom methylene spacer between the backbone and recognition moieties, gave a good modeling prediction for favorable base-pairing to a cornplementray polynucleotide.
  • Analogous structures containing a two-carbon spacer showed acceptable, but less favorable, binding conformation.
  • the structures shown in the figure, and the analogous two-carbon spacer structures, are preferred acylcic backbone moieties because of their general ease of synthesis. It should be mentioned, however, that a number of other backbone moieties are also entirely feasible and suitable -- although generally not as easy and/or inexpensive to synthesize.
  • the linkage or spacer connecting the backbone and recognition moieties in the polymer subunit must meet certain design criteria which are effective to position the recognition moieties for base-pair binding to the polynucleotide bases.
  • the linkage or spacer connecting the backbone and recognition moieties in the polymer subunit must meet certain design criteria which are effective to position the recognition moieties for base-pair binding to the polynucleotide bases.
  • the cyclic backbone moieties shown in Figure 3 modeling studies indicate that the most favorable binding conformation in the polymer occurs when the recognition moiety is attached directly to the 1' carbon of the ribose or ribose-like structures, and to the analogous 1' position of the morpholino structures. That is, the moiety is attached at normal point of attachment and with the normal stereoisomeric configuration of purine or pyrimidine bases to the ribose or deoxyribose groups of nucleosides.
  • one-and two-carbon atom spacers are required for placing
  • all of the backbone/recognition moiety linkages be either of define chirality or achiral.
  • the linkages are considered to be of defined chirality, for purposes of definition herein, if each of the linkages in any given subunit position in all of the polymer molecules has the same stereoisomeric configuration, or chirality. That is, although the subunits in different sequence positions may have different internal stereoisomer configurations (including achirality at specific sequence positions), all of the linkages at a given sequence position among the polymer molecules have the same stereoisomeric configuration. Usually defined chirality is most easily achieved by using the same stereoisomeric configuration in all of the subunits.
  • Subunits of this type are also readily synthesized, as will be discussed below, using natural D-nucleoside starting material. With reference to Figure 4, it is seen that only structures I and L have chiral linkages between the backbone and recognition moieties. These subunits are readily synthesized in homochiral form by using homochiral starting material , as will be described.
  • the one-atom linkages are attached to backbone nitrogen atoms, are are therfore achiral. Also, of course, the methylene spacers are themselves achiral.
  • Base-protected nucleosides are also useful starting reagents in a number of the subunit synthesis reactions to be described below. Methods for base protecting a number of the more common ribo- and deoxynucleosides from Example I are illustrated in Example 2. The methods detailed in the example are generally applicable for forming nucleosides with amine-protective groups.
  • nucleophile-insensitive intersubunit linkages include carbamate, amide, and sulfonamide linkages.
  • the corresponding nucleophile-sensitive protective groups for the recognition moieties include: benzoyl for the N4 of C; benzoyl or p-nitrobenzoyl for the N6 of A; acetyl or isobutyryl for the N2 of G; and N2,N6-bisisobutyryl for 2,6-diaminopurine residues. Removal of these groups after completion of polymer assembly is effected by treatment with ammonium hydroxide.
  • intersubunit linkage to be used is sensitive to nucleophiles, such as ammonium hydroxide
  • suitable protective groups are those which can be removed by strong non-nucleophilic bases -- via a ⁇ elimination mechanism.
  • nucleophile-sensitive intersubunit linkages include: carbonate; ester; and, to a lesser extent, triocarbamate linkages.
  • Suitable protective groups for the recognition moieties removable via ⁇ elimination include: 2-(4-nitrophenyl)ethoxy carbonyl or 2-(phenyl sulfonyl) ethoxycarbonyl for both the N4 of C and the N6 of A; and the 9-fluorenyl methoxycarbonyl for the N2 of G and the N2 and N6 of 2,6-diaminopurine residues. Removal of these groups after completion of polymer assembly is effected by treatment with the strong nonucleophilic base 1,8-diazabicyclo[5.4.0]u ⁇ dec-7-ene (DBU), under stringently anhydrous conditions.
  • DBU strong nonucleophilic base 1,8-diazabicyclo[5.4.0]u ⁇ dec-7-ene
  • the following backbone-protecting groups are preferred: for primary hydroxyls, the di(p-methoxy) trityl group; for primary amines, p-methoxytrityl; and for a secondary amine (as in morphilino-type backbone moieties), the phenylisopropoxycarbonyl group.
  • These protective groups can be readily removed by treatment with 0.2 M dichloroacetic acid in dichloromethane.
  • Subunits having a deoxydeoxynucleoside subunit structure can be obtained from commercial sources or prepared via literature methods, as described in Example I.
  • the subunits include the following ribosides and deoxyribosides, which are identified according to the struture numbers of the recognition moieties given in Figure 1: adenosine and deoxyadenosine (structure 1); 2,6-diaminopurine riboside and deoxyriboside (structure 2); cytodine and deoxycytodine (structure 3); 4-methoxy-2-pyrimidinone deoxyriboside (structure 4); 2-hydroxy-5-methyl pyrimidine deoxiriboside (structure 5); 2-hydroxypyrimidine riboside (structure 6); guanosine and deoxyguanosine (structure 7); inosine and deoxyinosine (structure 8); thioguanosine and deoxythioguanosine (structure 9); uradine and deoxyuridine (structure 10); th
  • the 5'-amino-2',5'-dideoxyribonucleosides (structure B in Figure 3) are prepared according to methods detailed in Example 3. Briefly, the selected deoxyribonucleosides, base-protected if necessary, are reacted with triphenyl phosphine, carbon tetrabromide, and lithium azide to form the corresponding 5 '-azidonucleoside, which is then reduced by hydrogenation in the presence of a palladium on carbon catylyst.
  • the nucleosides may be obtained as in Example 1, and base-protected as in Example 2. The stereochemistry of the reactant nucleosides is preserved in forming the 5' amino nucleoside analogs.
  • An alternative reduction method is used in reducing the azide group of 5'-azido-5-bromo uridine, as described in Example 3.2.
  • non-catalytic hydrogenation is needed to prevent removal of the ring bromine atom.
  • the azide is placed on the 5' position by first tosylating the 5' hydroxyl group, then displacing with azide, as detailed in Example 3.2.
  • the 3'-amino-2',3'-dideoxyribosnucleosides are prepared according to methods detailed in Example 4. Briefly, thymidine which is protected at its 5' hydroxyl is tosylated at its 3' hydroxyl, and this is followed by an intramolecular displacement involving the 2 oxy base substituent. The resulting ring is now opened by treatment with azide, yielding a 3' azido analog of the correct stereoisomeric form. This analog can be be converted to a selected-base analog by reacting the azide compound with a selected purine or pyrimidine base. The latter may be base protected, if necessary, for subsequent subunit coupling reactions, as described below. The thymidine or other azide analog is then reduced to produce the desired 3' amine nucleoside. The stereochemistry is of the thymidine starting material is preserved in the synthesis.
  • morpholino-type subunit derivatives represented by structure D in Figure 3 are detailed in Example 5 for a variety of different recognition moieties. Briefly, a selected nucleoside, base-protected if necessary, is dissolved in an ammonium salt, such as ammonium biborate, and then reacted with sodium periodate to form transient 2', 3' dialdehydes, which then close upon an ammonium ion to form morpholino ring having 2' and 4' hydroxyls. The compound is then treated with sodium cyanoborohydride to remove the ring hydroxyls. The ring nitrogen is preferably protected as a 2-phenylisopropylcarbamate for subsequent subunit coupling. The sterochemistry of the nucleoside starting material is retained.
  • an ammonium salt such as ammonium biborate
  • Deoxyribose subunit structures having a 5' acetic acid group can be prepared according the general synthetic scheme described in Example 6, which details the synthesis of the 5' acetic acid compounds represented by structure F.
  • a selected deoxyribonucleoside which is protected at the 3' hydroxyl is converted to the 5' aldehyde, and subsequently treated with a 2-carbon Wittig reagent to give the unsaturated acetic acid derivative.
  • Reduction of the side-chain olefin gives the desired 5' acetic acid compound.
  • the reaction preserves the sterochemistry of the starting nucleoside material
  • the analogous 5' formate compound (structure E in Figure 3) is formed by treating the above 5' nucleoside aldehyde with a one-carbon Wittig reagent, and hydroslysing the resulting enol to form form the corresponding formaldehyde derivative, which is oxidized to the desired acid.
  • the reaction preserves the stereochemistry of the starting nucleoside material.
  • the 5-atorn chain amino acid subunit represented by Structure I in Figure 4 is prepared according to the general procedures outlined in Examples 7-9. Briefly, an (S)-5 carboxy pyrrolidone was converted to the corresponding stereochernically pure
  • the purine used in subunit synthesis is preferably contains an electron-withdrawing group at the 6 position, to reduce the reactivity of the 1 and 3 ring nitrogens, i.e., to minimize pyrrolidone coupling at a ring nitrogen.
  • the pyrrolidone-derivized purine or pyrimidine may then be converted to the amine derivative, and base protected, if necessary, prior to the ring opening step which will be described below.
  • Example 7.1 and 7.2 detail methods for forming the cytosine pyrrolidone and its base-protected derivative.
  • the adenosine pyrrolidone formed in Examples 7.3-7.5 employs 6-chloropurine as the starting material, and the corresponding pyrrolidone is converted to the adenosine derivative through azide formation as described.
  • the adenosine is base-protected, as in Example 7.6, prior to ring opening.
  • a similar approach is used to generate base protected guanine pyrrolidone, starting from 2-amino-6 chloropurine, as described in Examples 7.7-7.9.
  • Similar methods are used in synthesizing 2,6-diaminopurine pyrrolidone, inosine pyrrolidone, and 2-hydroxypyrirnidine pyrrolidone, also as detailed in Example 7.
  • the pyrrolidone is then treated via a series of reactions which cleave the pyrrolidone ring to form an amino acid with a t-BOC protected amine.
  • Examples 8.1-8.4 describe the synthesis of such t-BOC amino acids having one of a number of selected recognition moieties.
  • the t-BOC protective group may be removed to form the corresponding amino acid represented by structure I in Figure 4.
  • the amino acid may be used directly for subunit coupling, according to reactions given below.
  • the t-BOC protected compound may be activated at its acid group for use in subunit coupling reactions.
  • a 6-member heterocycle such as a pyrimidine or nicotinate
  • a reactive methylene on the carbon ring position at which attachment to the backbone is desired.
  • the cytosine analog is formed by reacting 5-amino-5-bromopyrimidine to form the corresponding 5-position carboxaldehyde.
  • the uridine analog is formed by converting 6-hydroxynicotinic esters to the corresponding benzylic alcohols. This compound is converted to the aldehyde by reaction in the presence of manganese dioxide.
  • the aldehyde compound is then reacted with the desired amino acids, such as 4-aminobutyric acid, with reaction at the amine group producing an unstable imine which is reduced to form the stable amine subunit.
  • the secondary amine can be protected, for subunit coupling involving activation of the acid group, by a tBOC group as described in Example 10.1.
  • the C-carbon backbone analog of the above compound is prepared, using 3-aminoproprionic acid, and this compound is further treated with nitrous oxide to form the corresponding N-nitroso compound, which is subsequently reduced with H 2 IPd in the presence of iron salts to give the hydrazino acid subunit.
  • the coupling reactions used in forming the polymer molecules of the invention are stepwise reactions which join one selected subunit or subunit sequence to another selected subunit or subunit sequence.
  • the coupling reactions will be described generally with respect to coupling a single subunit B-R 1 having a selected recognition moiety R 1 to another single subunit B-R 2 having the same or a different selected recognition moiety R 2 , to form a dimer of the form R 1 R 2
  • N 1 and N 2 are nucleophilic backbone groups, such as hydroxyl and amine groups, which can be activated with an electrophile E, to form an activatedN 1 -E or N 2 -E backbone group which can then react with a second N 1 ----N 2 type backbone moiety to form an N 1 ----N 2 -E-N 1 ----N 2 backbone-linked dimer.
  • the subunit backbones of this types which have been described specifically above are the cyclic backbone structures A-D in Figure 3.
  • the activated N 2 nucleophile is the 3' hydroxyl group in structure C is the 5' hydroxyl and in structure D, the
  • a subunit having a selected recognition moiety R 1 is activated with an electrophile E.
  • the star at the recognition moiety indicates any required base protection.
  • the selected subunit is protected at its N 1 nucleophile, to ensure that (a) only the N 2 nucleophile is activated and (b) the activated subunit cannot self-polymerize.
  • the protective group is preferably an acid-labile group, such as dimethoxytrityl (DMTO) .
  • DMTO dimethoxytrityl
  • the activating reagent shown in Figure 5A has the general form:
  • X oxygen or sulfur
  • C is an active electrophile capable of reacting with a nucleophile, such as a hydroxyl oxygen or amine nitrogen, with displacement of Y, to form the activated subunit:
  • Subunit activation reactions involving carbonyl activated subunits are detailed in Example 11 for the 5'-protected, 2' deoxynucleosides represented at A in Figure 4; in Example 12 for the 5'-protected amino nucleosides represented at B in Figure 4; and in Example 13 for the N-protected morpholino-type subunits shown at D in Figure 4.
  • the general reaction conditions used to activate the hydroxyl groups in these structures are generally applicable; subunit activation reactions involving thiocarbonyl-activated subunits are de tailed in Example 14 for the 5'-amino nucleosides represented at B; and in Example 15, for the morpholino-type subunits shown at D in Figure 3.
  • the activated complex is purified by conventional methods, such as silica gel chromoatography, and then reacted with a second subunit whose selected recognition moiety R 2 will form the next in-sequence recognition moiety in the completed polymer.
  • the coupling reaction is preferably carried out under mild conditions in which the activated group can react with backbone N 2 amine groups, but not N 1 hydroxyl groups. Therefore, the method is suitable for coupling subunits of the type represented by structures B-D in Figure 4, but not subunit structure A.
  • the oligorner can be extended by repeating the above steps of (a) activating the free N 2 nucleophile in the second subunit, separating the activated species from the activating agent, and coupling the activated compound with the next-in-sequence subunit, whose backbone is unprotected.
  • This method is used particularly in forming short oligorner blocks by solution methods which will be described below and which are suitable for solid-phase block assembly.
  • the second coupling method outlined in Figure 5B is much preferred.
  • the second method differs from the first method in that polymer growth occurs by addition of an excess of activated subunit to an existing subunit or polymer chain, rather than, as in the first method, by addition of an unactivated subunit to an activated chain.
  • the existing subunit or subunit chain is shown by a subunit whose recognition moiety is R 1 (second line in Figure 6B).
  • This subunit has a free N 1 backbone nucleophile and an N 2 nucleophile which is protected by a preferably acid-stable linkage to a solid support or by virtue of being linked to a chain of subunits which are linked to a solid support.
  • the coupling reaction adds the N 2 -activated second subunit to the N 1 -protected first subunit to couple the two through an N 2 -E-N 1 bond, and form a compound which is protected at both free backbone nucleophile sites.
  • This compound is now treated, for example by reaction with acid, to deprotect the acid-labile N 1 protective group on last-added subunit, and the procedure is repeated to build up a desired sequence polymer.
  • N 2 -activated subunit which will form the last-in-sequence subunit must be protected at its N 1 backbone site, to allow selective activation at the N 2 site and to prevent self-polymerization of the activated compound
  • the N 1 -deprotected subunit which is to couple to the activated subunit should be protected at its N 2 site to allow selective reaction of its N 1 moiety with the N 2 -activated subunit. Therfore, since the reacting subunits are both protected at one backbone site, the method is suitable for coupling nucleosides (structure A in Figure 4) as well as subunits whose cyclic backbones contain both amine and hydroxyl backbone nucleophiles, such as structures B-D in this figure.
  • Example 12 The reaction methods used in coupling structure A subunits through a carbonate bond are detailed in Example 12, Briefly, a subunit containing a 5' protected backbone moiety is activated at its 3' hydroxyl, and reacted with another subunit (or growing chain) which is protected at its 3' hydroxyl. The coupling reaction is carried out in the presence of a catalyst, such as N-methylimidazole or N,N-dimethylaminopyridine, which is necessary for forming the carbonate bond.
  • a catalyst such as N-methylimidazole or N,N-dimethylaminopyridine
  • this second coupling method for forming a polymer by solid-phase subunit addition, is that a substantial molar exess of the activated subunit can be added to the growing support-bound polymer at each polymer-addition step, to achieve subunit coupling to a very high percentage of the support-bound polymers. This insures that a large percentage of the support-bound polymers will contain the complete sequence of subunits desired.
  • the efficiency of subunit addition is limited by the efficiency of the activations steps.
  • Figure 6 shows the dimeric structures produced by coupling the cyclic backbone subunits indicated at A-A through D-D in Figure 4.
  • all of the subunit linkages are uncharged and achiral, i.e., do not contain chiral centers.
  • the linkages are stable in aqueous medium, as judged by the ability of polymers to resist hydrolysis over an extended period in neutral aqueous solution.
  • the structures, when joined to form polymers show acceptible Watson/Crick base pairing with complementary polynucleotides.
  • polymers formed from the subunits are stereoregular. This is achieved in the structures shown by (a) using natural nucleosides or nucleoside derivatives or synthetic nucleosides having the natural stereoisomeric configuration as subunits, and (b) joining the subunits by achiral intersubunit linkages. Exemplary coupling methods are detailed in Examples 11-15.
  • N 1 is a nucleophilic backbone group, such as a hydroxyl or amine group
  • E is an electrophile, such as a carboxyl or sulfonyl group, which, when activated, can react with a second
  • N 1 -----E type backbone to form an N 1 -----E-N 1 -----E backbone-linked dimer.
  • the subunit backbones of this types which have been described specifically above are the cyclic backbone struct ⁇ pes E-H in Figure 3, and the acyclic backbone structures I-N in Figure 4.
  • the N 1 nucleophile is the
  • E group in all of the cyclic structures is the carboxyl or corresponding sulfonyl group attached to the 5' position of the ribose ring, or to the 6' position of the morpholino ring.
  • N 1 and E groups are respectively, the backbone amine or hydrazine and carboxylic or sulfonic acid groups on the backbone moiety ends
  • the first coupling method which is illustrated in Figure 7A, is analogous to the method described above with reference to Figure 5A, where a first
  • N 1 -protected subunit is activated, then coupled directly to a backbone-unprotected subunit which forms the next-in-sequence subunit in the growing polymer.
  • the P 1 protective group on the subunit N 1 is preferably an acid-labile protective group, such as t-butoxycarbonyl or a clevable linker bound to a solid support.
  • the backbone N 1 site may be protected by its attachment to a solid support, with subunit addition to the growing polymer occuring via the activated E electrophile of the last-added subunit.
  • the activation reaction is designed to yield an activated or chain-terminal moiety of the form N 1 -----E-X, where X is as described above with reference to Figure 6, and the activated subunit has much the same reactivity toward backbone nucleophilic groups as does the activated subunit in the previously described method. That is, the activation is designed to produce much the same active coupling group as in the
  • N 1 ----N 2 type backbones but where the reactive carbonyl or sulfonyl group is provided by the backbone itself rather than by a carbonyl or sulfonyl activating reagent, as in the methods shown in Figure 6.
  • the activation can be performed readily by reacting the N 1 ----E type subunit with a carbodimide in the presence of p-nitrophenol, where the backbone electrophile is a carbonyl group, or by reacting with a carbodiimide in the presence of imidazole, 1,2,4 triazole or tetrazole, where the backbone is a sulfonyl.
  • Subunit activation reactions involving carbonyl ele ctrophili c groups are detailed in Examples 16 and 17.
  • the general reaction conditions used to activate the N-amino-morphilino and linear chain backbone carbonyl groups are generally applicable to the other N 1 ----E type backbone structures shown in
  • the activated complex is purified by conventional methods, such as silica chromatography, or, in the case of a support-bound activated chain, simply washed, and then reacted with a second subunit whose selected recognition moiety R 2 will form the next in-sequence recognition moiety in the completed polymer.
  • the coupling reaction yields a dimer whose subunits are therefore coupled through an E-N 1 bond, as indicated in the figure.
  • both subunits must be suitably base protected during the activation and coupling reactions.
  • the polymer can be extended by repeating the above steps of (a) activating the free E electrophile in the second subunit, (b) separating the activated species from the activating agent, and (c) coupling the activated compound with the next-in-sequence subunit, whose backbone is unprotected.
  • the second coupling method which is illustrated in Figure 7B, is directly analogous to the method described above with respect to Figure 5B.
  • a first subunit having an E-protected backbone is reacted with a second subunit with an activated E group and a protected N 1 nucleophile, to form a dimer linked through an E-N 1 bond and protected at both free backbone end groups.
  • the P 2 protective "group” takes the form of an acid-stable linkeage to a solid support.
  • the N 1 -protected subunit is prepared and activated as above. Coupling conditions generally follow those used in the above cyclic subunit coupling reactions.
  • n 1 -protected and E-unprotected subunits are reacted together in the presence of a suitable E-activating agent, such as a carbodiirnide, as indicated
  • Figure 8 shows the dimeric structures produced by coupling the cyclic amd acyclic backbone subunits indicated at E-K in Figures 3 and 4, and indicated at E-E through K-K, repsectively.
  • the nucleosides subunits in structure F-F in Figure 3 are joined by an ester linkage.
  • the structures, when joined to form polymers show Watson/Crick base pairing with complementary polynucleotides.
  • N 1 -----N 2 backbone subunits As with the N 1 -----N 2 backbone subunits described above, all of the N 1 ----E backbone structures shown are stereoregular. This is due to (a) the homochiral (structures E-H in Figure 3 and I and L in Figure 4) or achiral (structures J, K, M, and N in Figure 4) nature of the recognition moiety attachment to the backbone moiety, and (b) the achiral subunit linkage.
  • achiral subunit linkages it may be desirable to introduce one or more charged achiral subunit linkages in the polymer molecules.
  • the backbone charge may be used to enhance polymer solubility in many solution applications or to adjust polymer-target binding constants, as will be described further below.
  • Polymers having regularly spaced charged achiral backbone linkages, e.g., between every second or third subunit, are suitable for the novel diagnostic system which is described below.
  • polymeric units composed of two or more subunits which are internally joined by achiral uncharged linkages are to be linked by a charged achiral linkage. These uncharged units are formed by one of the methods described in Sections A and B above.
  • the dimer subunits which are to be linked are selected such that that the free N 2 group of one dimer can be coupled to the free N 1 group of the other dimer through a suitable charged achiral linkage.
  • the preferred linkage is a phosphodiester linkage which requires that the free N 2 group on one subunit and the free N 1 group on the other subunit both be hydroxyls. As can be seen in Figure 3, this condition is met if dimers formed from either structure A or structure B subunits, which both have a free N 2 hydroxyl, are linked to dimers formed from structure A, C, or D subunits which all have a free N 1 hydroxyl.
  • dimer A which is protected at its N 1 position, as indicated, is carried through a series of reactions which lead to an reactive phosphoester coupling species linked to the N 2 hydroxyl through a phosphoester bond, as indicated in Figure 9.
  • the preferred coupling method which is detailed in U.S. patent application for "Polynucleotide Assay Reagent and Method", Serial Number 712,396, filed March 15, 1985, and which follows literature methods for forming phosphodiester bonds between base-protected nucleosides or nucleoside analogue subunits, involves formation of a reactive N 1 protected N 2 -(p-chlorophenyl2-cyanoethyl)-phosphate. The activated dimer is then reacted with the second dimer to form a tetramer which is linked alternately by uncharged and charged achiral intersubunit linkages.
  • the phosphodiester linkage method may be incorporated, either is solution or support-type polymer synthesis method, according to the general procedures outlined above for polymer coupling involving achiral, but uncharged linkage reactions.
  • solubility enhancement is generally achieved with 1-3 charged backbone linkages per polymer of about 20 subunits, and preferably no more than about one charged linkage per four subunits.
  • achiral backbone linkages are considered substantially uncharged if they contain less than about one charge backbone linkage per dimeric unit, and preferably one or fewer backbone linkage per tetrameric unit.
  • the assay system includes a solid support reagent composed of a multiple polynucleotide-binding polymers which are constructed according to the invention linked to a solid support.
  • the polymers are constructed, according to targeting strategies detailed below, for binding specifically to target polynucleotide analyte at a selected melting temperature (T m ).
  • the sytstem also includes polycationic reporter molecules which are designed to bind to the fully charged analyte backbone, but not the uncharged or only partially charged polymer backbone, under selected binding conditions.
  • Each reporter molecules carries one or more reporter groups, and each polynucleotide can accomodate binding of typically several thousand or moe reporter molecules.
  • the system has an ampliication factor, in terms of reporter signal per bound analyte molecule, of several orders or magnitude.
  • the assay system and its method of practice are more fully described in co-owned U.S. Patent application for "Polynucleotide Assay Reagent and Method" , Serial No. 712,396, filed March 15, 1985.
  • sequence-specific polynucleotide binding polymers having achiral predominatly uncharges intersubunit linkages for diagnostic applications derives from two factors.
  • their target binding is relatively insensitive to salt and so the polymer/target annealing step can be carried out under low salt conditions wherein there is no competitive annealing between the target and complementary nucleic acid sequences in the solution.
  • the polymer's reduced charge or uncharged backbone allows use of the above-described reporter molecules which are designed at high density to the target analyte's polanionic backbone.
  • the design considerations applied in preparing a polynucleotide binding polymer for use as a diagnostic reagent are governed by the nature of the target analyte and the reaction conditions under which the analyte is to be assayed.
  • a first consideration there is selected a non-homopolymeric target base sequence against which the polymer is directed.
  • This target sequence is preferably unique to the analyte being assayed, i.e., is calculated to occur only with some defined small probability (such as 1% or less) in an assay mixture containing a given number of unique-sequence bases.
  • P that a 9-base target sequence will be found in a 20 kilobase polynucleotide is about 20x10 3 /2x10 5 or
  • the probability that a 16-base target sequence will be present is about 20x10 3 /4.3x10 9 or
  • a polymer having 9-16 recognition moieties specific for a defined 9-16 base target sequence should have high specificity for the target sequence in an assay mixture containing only viral genomes, whose greatest complexities correspond to about 400K of unique-sequence bases.
  • the polymer/analyte binding affinity, and particularly the temperature at which the polymer just binds with the target sequence can be selectively varied according to (a) polymer length, (b) the number of hydrogen bonds that can be formed between the recognition moieties and the corresponding, in-sequence bases of the analyte target sequence, (c) backbone charge density, and (d) a concentration of denaturants, such as formamide, which reduces the temperature of melting.
  • the melting temperature of oligonucleotide duplexes in the 10 to 20 bp range increases roughly 3°C per additional base pair where the complementary bases are capable of forming two hydrogen bonds, and about 6°C per additional base pair where the complementary bases are capable of forming three hydrogen bonds. Therefore, the length of a target sequence, which is initially selected to insure high binding specificity with the polymer, may be extended to achieve a desired melting temperature with the complementary-base polymer, under selected assay conditions.
  • the target sequence may be selected to have a high percentage of guanine plus cytosine bases for achieving a relatively high polymer/analyte melting temperature, or a relatively high percentage of adenine plus thymine bases, for achieving a relatively low melting temperature.
  • the backbone charge density of the polymer must be substantially less than that of the polynucleotide analyte, to allow preferential binding of polycationic reporter molecules to the analyte, under conditions where reporter binding to the polymer does not occur. This requirement is met where the spacing between the adjacent negative charges in the polymer backbone is at least twice as great as the spacing between adjacent charged phosphodiester linkages in the analyte.
  • the charge density of the backbone also has an important effect on the polymer/analyte melting temperature, particularly under low salt conditions, where any charge-charge repulsion between the analyte and polymer backbones can act to lower the temperature at which the two can anneal.
  • a polymer having a less-charged backbone would be expected to show (1) a higher analyte/polymer melting temperature, and (2) less dependence of the melting temperature on salt concentration.
  • an uncharged polymer such as an all-carbamate-linked or all amide-linked polymer, will allow the analyte/polymer annealing reaction in the diagnostic method to be carried out under a wider range of salt-concentrations than a partially charged polymer, wherein some of the linkages constitute achiral but charged phosphodiester linkages.
  • sequence-specific gene inactivating agents should only effect inactivation of a targeted genetic sequence which contains an amount of sequence information adequate to assure a high probability of that target sequence being unique to the pathogenic state.
  • an agent which can effect target inactivation by binding to only three or four contiguous bases of its target sequence would invariably also attack many mRNAs required for normal cellular functions.
  • an agent which requires binding to 30 or more bases in order to effect target inactivation would have an exceedingly small probability of also attacking non-target messengers involved in normal cellular functions.
  • a reasonable estimation of the desired amount of sequence information which a gene-inactivating agent should recognize when that agent is targeted against a disease-specific mRNA can be calculated as follows.
  • the human genome contains roughly 5 billion base-pairs of unique-sequence DNA. However, essentially all of this material exists in the duplex state and so is largely unavailable for binding to single-strand-directed polynucleotide-binding agent. Thus, polymer binding is largely restricted to RNA transcripts of the genomic DNA. Of that genomic DNA, something on the order of 1% is transcribed in any given cell type.
  • MIL Minimum Inactivation Length
  • a sueful concept in this regard is that of a minimum inactivating length (MIL), which we define as the minimum number of contiguous bases of a target sequence to which an agent must bind in order to effect gene inactivation in vivo. Irrespective of whether or not the agnet binds a longer sequence of bases, it is this MIL value which determines the number of bases in the targer "recognized" by that agent -- which, in turn, determines the agent's level of sequence specificity.
  • MIL minimum inactivating length
  • a gene-inactivating agent has a 16-subunit length but an MIL value of only 10.
  • Such an agent would be expected to inactivate any genetic sequences containing any of the component 10-contiguous-base target sequences.
  • a cellular pool comprising 200 million bases of unique-sequence single-stranded genetic material, there are an estimated 1000 such target sequences.
  • the agent would also be expected to forma variety of partially mispaired complexes with sequences lacking even the required 10-contiguous-base target sequences -- with some of those rnispaired complexes having sufficient stability to effect inactivation of the participating nontarget genetic sequences.
  • the activity of the targeted gene will show a dramatic reduction at the subunit length corresponding to the MIL value. That is, the MIL value corresponds to that number of subunits just sufficient to block the activity of the targeted genetic sequence.
  • the minimum inactivating length is that minimum polymer subunit length which effects essentially complete blockage of new ⁇ -globin synthesis.
  • this MIL will vary at small amount depending on the targeted sequence, but in general this relatively simple extracellular method serves to give a quite good approximation of the intracellular MIL for any given backbone type.
  • the first three of the above methods actually comprise target selection procedures, while the latter two methods are effected during synthesis of the agents.
  • a difficulty with method (i) is that sequence specificity requirement generally preclude reduction of the agent's length to less than about 14 subunits.
  • the difficulty with methods (ii) and (iii) is that other more critical targeting criteria can so severely limit the optimal target region that these two MIL adjustment methods have little latitude in which to achieve a significant effect. Because of these limitations, most MIL adjustments are preferably achieved by methods (iv) and (v).
  • the polymer After selecting a desired polymer length and recognition moiety sequence, according to factors considered in Section IV, the polymer is assembled, using the general subunit coupling procedures detailed above.
  • One method of polymer assembly involves initial preparation of an appropriate set of dimers, linking selected dimers to form tetramers, linking of these to form octamers, and so on. This method is carried out in solution, substantially according to methods described with reference to Figures 5A and 7A above. It should be noted that all couplings need not necessarily be between oligomers of equal size.
  • each coupling product is roughly twice the mass of the precursors and so purification of the product of each coupling is a simplified matter.
  • Example 18 below details the assembly of an 8-subunit carbamate-linked polymer formed by this method.
  • One preferred method for polymer synthesis is stepwise assembly on a solid support.
  • the first subunit in the polymer is attached through its N 2 backbone group to a solid support.
  • a solid support such as glass beads derivatized with cleavable, preferably acid-stable, long-chain linkers are employed as the support material, and prepared for attachment of N 2 nucleophile, such as a 5' hydroxyl of a nucleoside, as descibed in Example 19.
  • the glass beads are reacted with a subunit which has a preferably acid-labile N 1 . protected group, and an activated N 2 or E backbone group, as detailed in Section III above.
  • any unreacted linker nucleophiles are capped by addition of a suitable capping reagent, such as p-nitrophenyl acetate, and thereafter the support is washed and filtered.
  • a suitable capping reagent such as p-nitrophenyl acetate
  • the protecting group on the N 1 terminal subunit is removed, typically by acid treatment, and after neutralization, the support is then reacted with an excess of the next-in-sequence subunit (or polymer unit) which is activated at its free N 2 backbone moiety. The excess of activated subunit maximizes the number of support-bound subunits which are chain-elongated.
  • one feature of the solid support assembly method is the need for high coupling efficiencies at each subunit addition step, and the concentration of added activated subunit is selected to maximize this efficiency. Chain elongation is continued in this manner, with capping of failure sequence, after each subunit addition, until the polymer of the desired length and sequence is achieved.
  • the general method is illustrated in Example 20 for the preparation of a 14-subunit carbonate-linked polymer, in Example 21, for the synthesis of a 19-subunit carbamate-linked polymer, and in Example 22, for the assembly of a 19-subunit amide-linked polymer.
  • the polymer may be capped with a suitable charged or uncharged group coupled to the terminal N 1 group.
  • the capping reagent is preferably a charged group which imparts an end terminal charge to the polymer.
  • the polymer is cleaved from the support, e.g., by treatment with a nucleophile, such as ammonium hydroxide, and the polymer is purified, such as by anion exchange chromatography.
  • the assembled, purified polymers are attached to a solid support, such as a cellulose support by known coupling methods, such as those detailed in Example 20.
  • a solid support such as a cellulose support by known coupling methods, such as those detailed in Example 20.
  • Examples 24, 26, and 28 are illustrative.
  • nucleosides are obtained from Sigma Chemical Co., St. Louis, MO: deoxyuridine, deoxyguanosine, thymidine, deoxyadenosine, deoxycytidine, 5-bromodeoxyuridine, deoxyinosine, 2,6-diamino-9-(2-deoxy- ⁇ -D-erythro-pentofuranosyl)9H-purin e (2,6-diaminopurine deoxyriboside), uridine, guanosine, 5-methyluridine, adenosine, cytidine, 5-bromouridine, inosine.
  • 2,6-Diamino-9-( ⁇ -D-ribofuranosyl)-9H-purine (2,6-diaminopurine riboside) is obtained from Pfaltz and Bauer, Inc., Division of Aceto Chemical Co., Inc., Waterbury, CT.
  • the following nucleosides are prepared by the literature methods indicated:
  • 1-(2-Deoxy- ⁇ -D-erythro-pentofuranosyl)-2-pyrimidinone (2-hydroxypyrimidine deoxyriboside) is prepared by the method of P. Kohler, E. Uolz, U. Sequin, and C. Tamm in Nucleic Acid Chemistry (L.B. Townsend and R.S. Tipson, eds) John Wiley and Sons, Inc. (1976).
  • 1-(2-Deoxy- ⁇ -D-erythro-pentofuranosyl)-4-methoxy-2-pyrimidinone is prepared by the following procedure: 1-(3',5'-Di-O-benzoyl-2-deoxy- ⁇ -D-erythro-pentofu ranosyl)-4-rnethylthio-2-pyrimidinone (prepared as in D. Cech and A. Holy, Collection of Czechoslov Chem Comm (1977) 42:2246) is treated with 200 mL of 0.2M sodium methoxide solution. The resulting solution is allowed to stand overnight at room temperature. This solution is then neutralized with Dowex 50X8 (H+ form) and filtered; the residue is dissolved in water. (100 mL) extracted with ether (2.50 mL) and the aqueous phase evaporated. The residue is an amorphous material which isused directly in succeeding reactions.
  • 2-Amino-9-(2-deoxy- ⁇ -D-erythro-pentofuranosyl)-1, 9-dihydro-6H-purine-6-thione (deoxythioguanosine) is prepared by the procedure of R.H. Iwamoto, E.M. Acton, and L. Goodman, J Med Chem (1963) 6:684.
  • 1-( ⁇ -D-Ribofuranosyl)-2-pyrimidinone (2-hydroxypyrimidine riboside) is prepared by the procedure of U. Niedballa and H. Uorbruggen, J Org Chem (1974) 39:3668.
  • 1-(2-Deoxy- ⁇ -D-ribofuranosyl)-4-methoxy-2-pyrimid inone (2-hydroxypyrimidine deoxyriboside) is prepared by the procedure of R. Wightman and D. Holy, Collection of Czechoslov Chem Comm (1973) 38: 1381.
  • 2-Arnino-9-( ⁇ -D-ribofuranosyl)-1,6-dihydro-6H-purine-6-thione is prepared by the procedure of J.J. Fox, I. Wempen, A. Hampton and I.L. Doerr, J Amer Chem Soc (1958) 80: 1669.
  • Dimethoxytrityl chloride, N-benzoyladenosine, N-benzoyl-2'-deoxyadenosine, N-benzoylcytidine, N-benzoyl-2'-deoxycytidine and N-isobutyryl-2'-deoxyguanosine are obtained from Sigma Chemicals, St. Louis, Missouri.
  • N-2 9-fluorenylmethoxycarbonyl derivative is prepared by the procedure below which is general for the protection of nucleoside amino groups:
  • 9-fluorenylmethoxycarbonyl chloride (5 mmol) is added and the solution maintained at room temperature for 3 hours.
  • the reaction is cooled in an ice bath and water (1 mL) is added. After stirring for 5 minutes cone. ammonia (1 mL) is added and the reaction stirred for 15 minutes.
  • the solution is evaporated to near dryness and the residue dissolved in chloroform (10 mL). This solution is washed with sodium bicarbonate solution (5 mL, 10%), dried over sodium sulfate and evaporated. The residue is coevaporated several times with toluene and the product chromatographed on silica gel using a gradient of methanol in methylene chloride (0-50%).
  • N-Isobutyrylguanosine is prepared by the method of Letsinger and Miller, J Amer Chem Soc (1969) 91:3356.
  • N-2 9-fluorenylmethoxycarbonyl derivative is prepared by the method of J. Heikkla and J. Chattopadhyaya, Acta Chem Scand (1983) B37:263.
  • the N-2 acetyl derivative is obtained from
  • N-6 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Hirnmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583.
  • N-6 4-Nitrobenzoyl-2'-deoxyadenosine is prepared using the procedure above for FMOC-guanosine except that 4-nitrobenzoyl chloride is substituted for FMOC chloride.
  • N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya, Tetrahedron Lett (1981) 22: 3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
  • N-6 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Hirnmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583.
  • N-6 4-Nitrobenzoyladenosine is prepared using the procedure above for FMOC-guanosine except that 4-nitrobenzoyl chloride is substituted for FMOC chloride.
  • N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya Tetrahedron Lett (1981) 22:3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
  • the N-4 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Himmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583.
  • the N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is, prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya Tetrahedron Lett (1981) 22:3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
  • the N-4 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Himmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24: 3583.
  • the N-6 2--(phenylsulfonyl)-ethoxy carbonyl derivative is prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya Tetrahedron Lett (1981) 22: 3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
  • N-2,N-6-bis (9-fluorenylmethoxycarbonyl) derivative of 2,6-diaminopurine riboside is prepared by the general procedure.
  • the N-2 ,N-6-bis (isobutyryl) derivative is prepared by the general procedure.
  • N-2 9-fluorenylmethoxycarbonyl derivative of 2'-deoxythioguanosine is prepared by the general procedure.
  • Carbon tetrabromide, sodium azide, P-toluenesulfonyl chloride (tosyl chloride), triphenyl phosphine and 10% palladium on carbon are purchased from Aldrich Chem Co. Lithium azide is obtained from Kodak Laboratory and Specialty Chemicals. 3.1 General Method
  • the nucleosides employed in this example are thymidine, N6-benzoyl-2'-deoxyadenosine, N4-benzoyl-2'-deoxycytodine, and 2'-deoxyinosine, 2-hydroxy-pyrimidine-2'-deoxyriboside and the N2-N6-bisisobutyryl derivative of 2,6-diamino purine-2'-deoxyriboside (see Example 1).
  • the required dried nucleoside (1 mmol) is weighed into a reaction vessel containing triphenyl phosphine (1.01 mmol) and lithium azide (5 mmol). After the solids are suspended in DMF, carbon tetrabromide (1.0 mmol) is added to the vessel.
  • the 5'-azido derivative of 5-bromo-2'-deoxy uridine is prepared via the method described above.
  • the 5'-azido-t-bromo-2'-deoxyuridine (1 mmol) is treated with triphenyl phosphine (1.5 mmol) in pyridine at room temperature for 2 h.
  • Concentrated ammonia (1 mL) is added to the reaction vessel. After 14 h the solvent is removed under vacuum. The residue is dissolved in THF and this solution is added to hexanes. The precipitate is collected and the solid triturated with the appropriate solvent to afford the 5'-amino-5-bromo-2'-deoxyuridine.
  • the resulting tosylate (1 mmol) is treated with sodium azide (6 mmol) in DMF at 80-100°C for a few h.
  • the solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with chlorofotrm/methanol solvent mixtures.
  • the azide is reduced to the desired amine using the above procedure.
  • Alternate protecting groups for the base nitrogens of 2'-deoxycytidine, 2'-deoxyadenosine, 2'-deoxyguanosine, and 2,6-diaminopurinedeoxyriboside are 2-(phenylsulfonyl) ethoxycarbonyl for 2'-deoxycytidine and 2'-deoxyadenosine and the use of the 9'-fluorenylmethoxycarbonyl (FMOC) to protect 2'-deoxyguanosine and 2,6-diaminopurine deoxyriboside, as in Example 2.
  • Example 4 Preparation of 3'-amino-2', 3' -dideoxyribonucleoside subunits. Thymidine is transformed to 5'-O-acetyl-3'-azido-2', 3'-dideoxythymidine and this is converted to 3'-azido-2', 3'-dideoxyadenosine and 3'-azido-2', 3'-dideoxyguanosine via the methods of M. Imazawa and F. Eckstein, J Org Chem (1978) 43:3044.
  • the base moiety of the 3'-azidoadenosine is protected as either the benzoyl or the 2-(phenylsulfonyl)ethoxycarbonyl derivative (as shown in Example 1).
  • N-2 of the 3'-azidoguanosine is protected as either the acetyl or FMOC derivative (see Example 1).
  • the reduction of these 3'-azido functions to the 3'-amino-2', 3'-dideoxyribonucleosides is performed as shown in Example 3.
  • Ammonium biborate, sodium m-periodate, sodium cyanoborohydride, uridine, N4-benzoyl cytidine, and N6-benzoyl adenosine are obtained from Sigma Chemical Co.
  • N2-isobutyryl guanosine is prepared by the general method of Letsinger and Miller (J Amer Chem Soc. (1969) 91:3356).
  • 2-Phenyl-2-propanol and bis (p-nitrophenyl) carbonate are obtained from Aldrich Chemical Co.
  • the morpholino derivative of uridine is prepared by dissolving 1 mmol of uridine plus 2 mmol of ammonium biborate, (NH 4 ) 2 B 2 O 7 , in 5 ml of water. While stirring in an ice bath protected from direct light 1.2 mmol of sodium m-periodate in 5 ml of water is added. After 90 min, 0.2 ml of 1,2-propanediol is added and stirring is continued for 10 min at rt. Thereafter, while stirring in a well-ventilated hood, 0.25 g of sodium cyanoborohydride dissolved in 3 ml of water is added and the mixture stirred for 4 hr at rt.
  • reaction mixture is reduced to an oil under vacuum in a warm water bath, resuspended in a minimal volume of methanol, layered on a silica gel chromatography column, and the column eluted with methanol/1% triethylamine.
  • the morpholino product elutes from the column as a sharp band moving significantly slower than the original ribonucleoside and its oxidation products.
  • the morpholino product has a UV spectrum essentially identical to the parent ribonucleoside, but its mass is lower by 17 daltons (determined by mass spectral analysis using fast atom bombardment activation).
  • the morpholino nitrogen is next protected by reacting the product with 2-phenylisopropyl phenyl carbonate.
  • the required active carbonate is prepared and reacted with the morpholino subunit essentially by the methods of Sandberg and Ragmarsson (Int J Peptide Protein Res (1974) 6:111).
  • the hydroxyl at the position corresponding to the original 5' can be activated by dissolving the N-protected subunit in a minimal volume of dry dimethylformamide and adding 2 equivalents of bis (p-nitrophenyl) carbonate plus 0.2 equivalent of triethylamine, N-methylimidazole, or N,N-dimethylamino pyridine. After 3 h at rt, the reactions mixture is reduced in volume under vacuum in a warm water bath.
  • the thick syrup is resuspended in a small volume of dichloromethane and layered on a silica gel column, which is subsequently eluted with a mixture of dichloromethane/ether (1:1 by vol) 0.2% in N,N-dimethylaniline by volume.
  • the activated product moves substantially slower than p-nitrophenol and bis (p-nitrophenyl) carbonate but much faster than the unreacted starting material.
  • the activated product is readily visualized on silica gel TLC plates simply by exposing to ammonia fumes, whereupon a yellow nitrophenolate ion is generated within one to two minutes.
  • ribonucleosides bases protected where necessary as in Example 2 are converted to their corresponding morpholino derivatives by essentially the same methods -- though varying amounts of methanol must be added to effect dissolution of the protected ribonucleosides before the initial periodate oxidation step.
  • the preferred base-protective groups for the common starting ribonucleosides are: the phenylsulfonylethoxycarbonyl moiety for cytidine and adenosine and the 9-fluorenyl methoxycarbonyl moiety for guanosine.
  • Triethylamine, pyridine/sulfur trioxide complex, p-nitrophenol, dicyclohexylcarbodiimide, and 40% aqueous dimethylamine are obtained from Aldrich.
  • the residue is dissolved in dry pyridine (10 mL) and treated with benzyloxycarbonylmethylenetriphenlyphosphorane (1.5 mmol) for 24 h at 37°C. The mixture is then evaporated to dryness under reduced pressure and the residue dissolved in methylene chloride, washed with dilute HCl and water, then the organic phases are dried over sodium sulfate and solvent removed under reduced pressure. The residue is chromatographed on silica using a gradient of methanol in chloroform (0-20%).
  • the unsaturated ester (1 mmol) from the previous paragraph is dissolved in ethyl acetate/ethanol (40 mL, 1/1, v/v) containing cyclohexadiene (5 mL) and 10% palladium on charcoal (100 mg) and the suspension is vigorously agitated under a nitrogen atmosphere for 5-24 h. The mixture is filtered and the solvents removed under reduced pressure. The residue is chromatographed on silica gel using a gradient of methanol in chloroform (5-50%). To a 0.2-0.5 M solution of the acid from the previous paragraph in ethyl acetate (or methylene chloride) p-nitrophenol is added in about 20% excess.
  • the calculated amount of dicyclohexylcarbodiimide is added to the solution at 0°C. After 0.5 h the mixture is allowed to come to room temperature and was held there for 1 h. The dicyclohexylurea which separated is filtered and washed with solvent. The combined filtrate and washings are evaporated to dryness under reduced pressure.
  • the ester may be used directly for coupling reactions or may be purified by short column chromatography on silica using an isopropanol in chloroform gradient (0-50%).
  • the active ester (1 mmol) is treated with 40% aqueous dimethylamine (2 mmol) in methanol to form the N,N-dimethylamide of the N-protected 3'-O-tert-butyl dimethylsilyl-2', 5'-dideoxyadenosine-5'-acetic acid.
  • the solvents are removed by evaporation under reduced pressure and the residue is desilylated by using the 2M HF/1M tert-butylammonium fluoride (TBAF) reagent described in B.L. Gaffney and R.A. Jones, Tetrahedron Lett (1982) 23: 2257.
  • TBAF 2M HF/1M tert-butylammonium fluoride
  • Potassium t-butoxide anisoyl chloride, 6-chloropurine, 10% palladium on charcoal, phthaloyl chloride, 2-amino-6-chloropurine, 20% aqueous tetraethylammonium hydroxide, 25% aqueous trimethylamine, 2-pyrimidinone, N-bromosuccinimide, trifluoroacetic acid, 4-nitrophenol, and N,N-disuccinimydyl carbonate are obtained from Aldrich. Lithium azide is obtained from Eastman Kodak, Rochester, New York. C18 reverse phase silica gel is obtained from Whatman, Hillsboro, Oregon.
  • Cytosine (2.2 mrnol) is dissolved in dimethyl sulfoxide (2 mL) which contained potassium tert-butoxide (2 mmol). This solution is added to a flask which contained (S)-5-(tosyloxymethyl)-2-pyrrolidone (1 mmol, prepared by the procedure of E. Hardegger and H. Ott, Helv Chim Acta (1955) 38:312). After dissolution, the mixture is stirred at 25°C for 6 h. The mixture is neutralized with acetic acid (2 mmol) and evaporated under reduced pressure. The residue is taken up in dimethyl formamide (5 mL) and evaporated under reduced pressure, and this procedure repeated three times.
  • This compound is prepared as for the alkylation of cytosine except that the alkylation is performed on
  • 6-chloropurine and the mixture was heated at 35-95°C for several h.
  • the mixture is cooled to room temperature, neutralized with acetic acid (2 mmol) and evaporated under reduced pressure, shaken with a mixture of 20% methanol/chloroform and 10% sodium bicarbonate.
  • the aqueous layer is washed with chloroform, the combined organic layers aree dried over sodium sulfate and evaporated under reduced pressure.
  • the residue is chromatographed on silica gel using a gradient of methanol in chloroform (0-25%).
  • the 6-chloropurine pyrrolidone (1 mmol) is treated with lithium azide (2 mmol) in dimethyl sulfoxide (2 mL) at 30-100°C for several h. At the end of this time the mixture is evaporated under reduced pressure, dissolved in chloroform, washed with 10% sodium bicarbonate, and the organic layer dried over sodium sulfate and concentrated under reduced pressure. The residue is chromatographed on silica using a gradient of methanol in chloroform (0-25%).
  • the azide from the previous example is hydrogenated by shaking the azide (1 mmol) with 30 pounds of hydrogen pressure in a solution of ethanol (10 mL) containing palladium on charcoal (100 mg, 10% by weight Pd) for 24 h. The solution is filtered through celite and the solvent evaporated. The residue is used directly for the next step.
  • the phthaloyl group is introduced at the N-6 position by the acylation of the adenosine pyrrolidone (1 mmol) with phthaloyl chloride (1.4 mmol) using the procedure above for the anisoylation of cytidine except that no ammonia was added in the workup.
  • the pyrrolidone tosylate is alkylated with 2-amino-6-chloropurine as per the preparation of the 6-chloropurine derivative. 7.8 Preparation of guanine pyrrolidone
  • the chloropurine pyrrolidone (1 mmol) from the previous paragraph is dissolved in diglyme (10 mL) and 25% aqueous trimethylamine (2 mL). The solution is stirred at rt for 5 h, water (10 mL) is added, and the mixture concentrated to 10 mL under reduced pressure. Acetic acid (2 mL) is added, and the mixture was evaporated under reduced pressure to an oil. The residue which contains tetraethylammonium acetate is used directly for the next step.
  • Guanine pyrrolidone (1 mmol) from the previous paragraph is reacted with acetic anhydride (5 mL) at reflux for 2 h.
  • the solvent is evaporated and the residue coevaporated with dimethylforrnarnide.
  • the residue is dissolved in methanol (5 mL) saturated with ammonia at 0°C, and the solution is stirred for 2 h at this temperature.
  • the solvent is evaporated under reduced pressure arid the residue purified by chromatography on a short column of silica using a gradient of methanol in chloroform (5-50%).
  • the acetyl group is introduced by the method above for N-2 acetylguanine pyrrolidone except that the methanolic ammonia treatment is carried out for 8 h.
  • the solvents are evaporated and the residue is phthaloylated as for the adenosine derivative.
  • This compound is obtained by the alkylation of 2-pyrirnidinone by the procedure used for the preparation of the 6-chloropurine derivative.
  • the general procedure described below for the cytosine pyrrolidone may be applied to all properly protected pyrrolidone derivatives.
  • the product, referred to as the t-BOC acid is formed by cleavage of the pyrrolidone ring to an acid and a t-BOC amine.
  • N-4-Anisoylcytosine pyrrolidone (1 mmol) is dissolved in methylene chloride (0.5 M solution) containing di-tert-butyl dicarbonate (2 mmol), triethylamine (1 mmol), and dimethylaminopyridine (1 mmol). The solution is stirred for 10 h at rt. The volatiles are removed and the residue purified on silica using a gradient of methanol in chloroform (0-20%). The residue is dissolved in tetrahydrofuran (0.2 M solution) to which is then added lithium hydroxide ( 3 mmol, as a 1 M solution).
  • cytosine t-BOC acid (1 mmol) is dissolved in aqueous acetic acid and treated with sodium nitrite
  • This compound is obtained from the uracil t-BOC acid by the following procedure: The uracil t-BOC acid (1 mmol) is dissolved in
  • the cytosine t-BOC acid (1 mmol) from the previous paragraph is dissolved in N-methylimidazole or pyridine (5 mL) and treated with chlorotrimethylsilane (5 mmol). After 15 min at room temperature the solution is treated with 2-(4-nitrophenyl)ethylchloroformate (3 mmol, obtained from F. Hirnmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583). The reaction iss maintained at room temperature for 8 h, then cooled in an ice bath, and water (1 mL) was added. After 5 min, 1 mL of concentrated ammonia is added and the mixture stirred at rt for 15 min.
  • the reaction is then evaporated to dryness and the residue dissolved in water.
  • the solution is made acidic with 2 M HCl and extracted with chloroform.
  • the combined organic extracts are dried over sodium sulfate and evaporated under reduced pressure.
  • the residue is purified by chromatography on silica using a gradient of methanol in chloroform (5-50%).
  • the product benzamide (25 mmole) is added to a 45 mL pressure vessel containing triethylamine (10 mL), benzene (10 mL), and bis(triphenylphosphine) palladium II chloride (0.375 rnmole).
  • the bomb is flushed with argon, sealed, and pressurized to 600 psi with carbon monoxide and then pressurized to 1200 psi with hydrogen.
  • the reaction vessel is heated in an oil bath with stirring at 145°C. after all gas absorption had ceased, the reaction is cooled, and the gases vented slowly. After addition of anhydrous ether, the reaction mixture is filtered and evaporated in vacuo.
  • the product was isolated following chromatography in silica gel using a gradient of isopropanol in methylene chloride (0-20%).
  • the 2-benzamidopyrimidine-5-carboxaldehyde (1 mmol) from the previous paragraph is dissolved in ethanol (5 mL) and treated with 4-aminobutyric acid (1 mmole) and triethylamine (1 mmol). After stirring for one hour, the, mixture is hydrogenated at 30 psi using 10% palladium on carbon (100 mg). After the absorption of hydrogen has ceased, the reaction is filtered and the solvents removed under reduced pressure. The residue is dissolved in methanol which is saturated with ammonia at 0°C and stirred at room temperature for 10 h.
  • the residue iss purified by chromatography on C18 silica gel using methanol water mixtures buffered to pH 7 with ammonium acetate (0.2 M).
  • the amino acid (1 mmol) from the previous paragraph is dissolved in ethanol (3 mL) containing di-tert-butyl dicarbonate (1 mmol).
  • Triethylamine (1.5 mmol) is added slowly at room temperature. After the evolution of carbon dioxide has ceased, the solvents are removed in vacuo and the residue used directly for the next reaction.
  • the t-BOC-amino acid from the previous paragraph is acylated on the exocyclic amino group by the general procedure in Example 2 using 2-(phenylsulfonyl)-ethoxycarbonyl chloride.
  • the product may be purified by chromatography on silica gel using a gradient of methanol in chloroform (0-50%).
  • Lithium borohydride is obtained from Aldrich.
  • Manganese dioxide is prepared as per Uereshchagin et al, Zhurnal Arganicheskoi Khimil, 8:1129 (1972).
  • Dowex resin is obtained from Bio-Rad Laboratories, Richmond, CA .
  • the benzylic alcohol (66 mmol) prepared in the previous paragraph is dissolved in ether (250 mL) and treated slowly (exothermic) with a slurry of manganese dioxide (54 g) in ether (100 mL). After twelve hours at room temperature, the mixture is filtered, the solvent removed under reduced pressure, and the residue distilled in vacuo to give 6-hydroxynicotinaldehyde.
  • the aldehyde from the previous paragraph is reacted with 4-amino butyric acid as for the preparation of the cytidine analog in Example 11.1
  • N-2 9-Fluorenylmethoxycarbonyl-2'- deoxyguanosine (1 mmol) is dissolved in anhydrous pyridine (2.0 mL) and kept at 0°C. Dimethoxytrityl chloride (1.2 minol) is added in portions (0.15 mmol) during an 8 hour period. After an additional 2 hours methanol (0.5 mL) was added and the solution is concentrated at reduced pressure. The residue is treated with a mixture of sodium bicarbonate (2 mL, 5%) and methylene chloride (5 mL). The aqueous layer is extracted with methylene chloride (2x5 mL) and the combined organic layers are dried over sodium sulfate and evaporated. The residue is coevaporated several times with toluene and the product is purified by chromatography on silica gel using a gradient of methanol in methylene chloride (0-10%).
  • the organic layer is washed twice with aqueous NaOH (5 mL, 0.1 M), water (2 mL), dried over sodium sulfate and evaporated.
  • the residue is applied to a silica gel column and eluted with a gradient of isopropanol in chloroform (0-10%).
  • the fractions containing the product are pooled, evaporated, dissolved in chloroform (10 mL) and the aqueous NaOH washes are repeated to remove traces of 4-nitrophenol.
  • the organic layer is washed with water, dried over sodium sulfate and evaporated.
  • the product is homogeneous by TLC and is used directly for preparation of the dimeric carbonates.
  • N-methylimidazole as the catalyst rather than N,N-dimethylaminopyridine.
  • the organic layer is evaporated to dryness under reduced pressure and purified by chromatography over silica gel with methylene chloride as eluant.
  • a solution of the 5'-O-dimethoxytrityl derivative from the previous paragraph (1 mmol) in methylene chloride (5 mmol) is added a solution of zinc bromide in methylene chloride (10 mL, 1 M) containing methanol (15 % v/v).
  • the reaction mixture is stirred for 30 min, quenched with ice, and extracted with methylene chloride.
  • the organic layer is washed with water, dried over sodium sulfate, and evaporated to dryness under reduced pressure.
  • the product is purified by chromatography over silica gel using a gradient of methanol in methylene chloride (0-10%).
  • the solvent is removed by evaporation under reduced pressure and the reaction allowed to sit overnight at rt.
  • the residue is dissolved in chloroform (20 mL) and washed with aqueous HCl (2 mL, 0.1 M), water (2 mL), aqueous NaOH (2 x 2 mL, 0.2 M), water (2 mL), and the organic layer dried over sodium sulfate and evaporated.
  • the residue is purified on silica gel using a gradient of isopropanol in chloroform (0-30%).
  • the isolated product is homogeneous on TLC and by 400 MHz NMR.
  • the residue is desilylated by using the 2 M
  • HF/1M tert-butylammonium fluoride (TBAF) reagent described in B.L. Gaffney and R.A. Jones, Tetrahedron Lett (1982) 23:2257.
  • TBAF HF/1M tert-butylammonium fluoride
  • the 3'-hydroxydinucleoside carbonates are converted to the 3'-(4-nitrophenyl)-carbonates by the method in Example 11. These derivatives may be reacted with the 5'-hydroxyl of a nucleoside or oligonucleoside carbonate to prepare higher order oligonucleoside carbonates.
  • Example 13 Formation of a carbamate intersubunit linkage.
  • Bis (p-nitrophenyl) carbonate, p-anisoyldiphenyl methyl chloride, N,N-dimethylaniline (DMA), and triethylamine are available from Aldrich Chemical Co.
  • the required 5'-amino-2', 5'-dideoxyribonucleoside (employing acetyl or benzoyl groups for base protection where necessary) (1 mmol) prepared in Example 3 above, is treated with p-anisoyl diphenylmethyl chloride (1.2 mmol) in pyridine. After 12 h the solvent is evaporated and the residue is redissolved in chloroform.
  • the 5'-tritylated aminonucleoside (1 mmol) is evaporated twice from DMF, then treated with bis-(p-nitrophenyl) carbonate (2 mmol) in the presence of triethylamine (or N,N-dimethylaminopyridine, or N-methyl ir ⁇ idazole) (catalytic amount) using DMF as the solvent. After 3 h, the solvent is evaporated and the residue dissolved in chloroform. This solution is washed twice with 0.01 N aqueous NaOH, once with water, then dried over Na 2 SO 4 .
  • the solvent is removed by rotovap and the residue is chromatographed on silica gel eluting first with a chloroform/0.1% DMA mixture, then with a chloroform/methanol/0.1% DMA solvent system. The appropriate fractions are combined and evaporated to dryness. The residue is dissolved in a minimum of THF and this solution is added to an excess of hexanes. The precipitated activated nucleoside is collected by filtration and dried under vacuum.
  • the required 5'-aminonucleoside (1.1 mmol), as prepared in Example 3 above, is twice evaporated from DMF.
  • the activated nucleoside (1 mmol) is added to the reaction vessel and the solids are dissolved in DMF.
  • the solution is concentrated to a small volume and allowed to stand overnight.
  • the solvent is completely removed under vacuum and the resulting residue is dissolved in chloroform.
  • This solution is twice washed with 0.01 N aqueous sodium hydroxide, once with water, then dried over solid sodium sulfate.
  • the solvent is removed by rotovap, and the residue is chromatographed on silica gel eluting with the appropriate methanol/chloroform/1% triethylamine solvent system.
  • the fractions containing the dimer nucleoside are combined, then evaporated to dryness.
  • the residue is dissolved in a minimum of THF and this solution is added to hexanes.
  • the precipitate is collected and dried under vacuum.
  • N,N-dimethylaminopyridine (DMAP) is purchased from Aldrich Chemical Co. Formation of the thiocarbamate intersubunit linkage of the 5'-aminonucleosides prepared in Example 3 is achieved in the same manner as described above in Example 13 with the following alterations in procedure.
  • the 5'-aminonucleosides employed have as base-protecting moieties either 2-(phenylsulfonyl)ethoxycarbonyl (for deoxycytidine and deoxyadenosine) or 9-FMOC (for guanosine and 2,6-diamino-2'-deoxyriboside) groups. The preparation of these molecules are described above (Example 3).
  • N-methylimidazole is purchased from Aldrich Chemical Co.
  • the solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with an appropriate chloroform/isopropanol/0.1% DMA mixture.
  • the fractions containing the desired activated nucleoside are combined, evaporated, and the resulting solid is dissolved in a minimum of THF.
  • the THF solution is added to an excess of hexanes and the resulting precipitate is collected and dried.
  • the chloroform solution is washed twice with 0.01 N aqueous NaOH, once with water, and then dried over Na 2 SO 4 .
  • the solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with chloroform/methanol solvent mixtures. The fractions containing the desired dimer are combined, then rotovaped to dryness.
  • the residue is dissolved in a minimum of THF and the solution is added to an excess of hexanes. The precipitate is collected and dried under vacuum.
  • Morpholino subunits linked by thiocarbamate moieties are prepared as related above in this example with the following experimental alterations.
  • the base-protecting groups are either
  • the coupling step employs this activated monomer (1 mrnol) with the requisite N'-free morpholino nucleoside (1.1 mmol) using DMAP or N-methyl imidazole as catalyst and DMF as the solvent while warming the solution to 35-40°C.
  • the 0.01 N aqueous NaOH washes are omitted, employing in their place water washes.
  • N,N-dimethylaminopyridine and N-methylimidazole are obtained from Aldrich.
  • the coupling of two subunits is performed by reaction of the 5'-N,N-acetamide-3'-hydroxy derivative with the with the 3'-silylated-5'-active ester. These are prepared as in Example 6.
  • the reagents (1 mmol each) are coevaporated several times with dimethylformamide, then dissolved in dimethylformamide (5 mL) and treated with N,N-dimethylaminopyridine (0.2 mmol) or N-methylimidazole (1 mmol).
  • the solvent is removed under reduced pressure and the reaction allowed to stand at room temperature for 2 h.
  • the residue is dissolved in chloroform and washed with dilute HCl, water, and the organic phase dried with sodium sulfate and solvents evaporated under reduced pressure.
  • the residue is purified by chromatography on silica gel using a gradient of methanol in chloroform.
  • the chain may be elongated by desilylation as in Example 6, and treatment of the resulting 3'-free hydroxy compound with the active ester as in the previous paragraph.
  • a fully protected t-BOC N-hydroxysuccinimidoyl ester or p-nitrophenyl ester prepared as in Example 11.3 and 11.4 (1 mmol) is dried by several coevaporations with dry dirnethyformamide.
  • the protected amino acid trifluoroacetate salt from Example 9 (1 mmol) is treated similarly.
  • the two components are separately dissolved in dry dimethylformamide (2 mL), mixed, and treated with diisopropylethylamine (1.0 mmol).
  • the solution is stirred at room temperature for 1 h, then the solvent is removed by evaporation under reduced pressure, the residue dissolved in chloroform and washed with dilute HCl.
  • the organic layer is dried over sodium sulfate, evaporated to dryness under reduced pressure, and the residue purified by chromatography on silica gel using a gradient of methanol in chloroform (5-50%).
  • the t-BOC dimer acid may be purified by chromatography on C18 reverse phase silica gel using water and methanol (trifluoroethanol) mixtures.
  • the t-BOC dimer active ester and the dimer acid trifluoroacetate are coupled to the t-BOC tetramer acid by the general procedure. Purification is best effected by chromatography on C18 reverse phase silica gel using water and methanol (or trifluoroethanol) mixtures buffered to pH 7.0 with triethylammonium acetate. In this manner chains of any length may be prepared by coupling an active ester with a free amine component.
  • DEAE cellulose is purchased from Sigma Chemical Co.
  • Preparative TLC plates are a product of EM Science purchased from UWR Scientific. HPLC equipment, columns, and supplies are available from Beckman Instruments, Inc.
  • the requisite carbamate-linked dimer (0.2 mmol) prepared as described in example 12, is treated with 4 mL of a 1/1/1 mixture of methanol/THF/glacial acetic acid at rt for 12 h. The solvent is removed under vacuum and the residue is taken up in a minimum of THF.
  • the THF solution is added to a large volume of hexanes and the resulting precipitate is collected and dried.
  • the acetate salt is dissolved in a 4/1 THF/ethanol mixture and DEAE cellulose (0.8 mmol of the base) is added to the vessel. After stirring for 20 min, the heterogeneous mixture is filtered and the filtrate evaporated to dryness. The residue is dissolved in a minimum of 4/1 THF/ethanol and this solution is added to hexanes. The solid is collected and dried. The precipitation procedure is repeated once.
  • N-5'-trityl dimer (0.1 mmol) (as prepared in Example 12) is evaporated twice from DMF, then treated with bis (p-nitrophenyl) carbonate (2 mmol) in the presence of triethylamine (or DMAP or N-methyl imidazole) (catalytic amount) using DMF as the solvent. after 3 h the solvent is evaporated and the residue dissolved in chloroform. This solution is washed twice with 0.01 N aqueous NaOH, once with water, then dried over Na 2 SO 4 . The solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with a chloroforin/isopropanol or methanol/0.1% DMA mixture.
  • the octamer is configured for further use as follows.
  • the N-5'-trityl octomer nucleoside is treated with 1/1/1 THF/methanol/glacial acetic acid and purified employing the conditions listed above.
  • the 5'-amino octomer nucleoside (0.01 mmol) is treated with succinic anhydride (0.02 mmol) in pyridine at room temperature for two hours.
  • the solvent is removed by rotovap and the residue is evaporated several times from ethanol.
  • the residue is taken up in 3/1 THF/ethanol and added to a 2/1 hexane/benzene mixture.
  • the solid is collected and dried.
  • N-5'-succinylated octamer nucleoside (0.01 mmol) is treated with a 1/1 mixture of pyridine/concentrated ammonia (1 mL). After standing overnight the solvent is removed by rotovap and the residue is evaporated from ethanol several times. The crude solid is purified using reverse phase (RP-18) HPLC chromatography. The elutants are evaporated and the residue is taken up in DMSO and precipitated from a 1/1 hexane/benzene solvent mixture. The solid is collected and dried.
  • Example 19 Preparation of a support-bound cleavable linker suitable for stepwise and stepwise/block assembly of polymers: activation of support and attachment of linker.
  • Long-chain alkylamine-derivatized controlled pore glass (Cat. No. 24875) is obtained from Pierce Chemical Co. 2,2-sulfonyldiethanol and dicyclohexylcarbodiimide are obtained from Aldrich Chemical Co.
  • One-tenth mol of 2,2'-sulfonyldiethanol (60 % by weight in water) is dried by mixing with three volumes of dimethylformamide (DMF), reducing to a thick syrup under vacuum in a warm water bath, adding a second quantity of DMF, reducing to a thick syrup, and repeating the process a third time. Dry DMF is then added to give a final sulfonyldiethanol concentration of 2 M.
  • DMF dimethylformamide
  • Example 20 Preparation of a 14-s ⁇ bunit carbonate-linked polymer targeted against a conserved seguencein the AIDS viral genome: stepwise assembly on a solid support: addition of the first subunit and chain extension.
  • N-methylimidazole, 4-(N,N-dirnethylamino) pyridine (DMAP), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are obtained from Aldrich Chemical co.
  • Methyl p-nitrophenyl carbonate is prepared from methyl chloroformate and p-nitrophenol.
  • the 2'-deoxycytidine subunit(0.1 mmol) prepared as in Example 4.2 (wherein the N4 carries a p-nitrophenethoxycarbonyl moiety, the 5' oxygen carries a di(p-rnethoxy)trityl moiety (DMT), and the 3' oxygen carries a p-nitrophenoxy carbonyl moiety) is dissolved in a minimal volume of dry tetrahydrofuran (THF) or dimethylformamide and added to 0.5 g of thoroughly dried controlled pore glass support carrying a cleavable linker, prepared as in Example 19.
  • THF dry tetrahydrofuran
  • Catalyst (either 1 mmol of N-methylimidazole or 0.1 mmol of DMAP) is introduced and the slurry is mixed by rocking or trembling (not stirring) for 2 hours. The slurry is next filtered, and the solid washed thoroughly with THF. Any unreacted hydroxyls are capped by adding two ml of THF 1 M in methyl p-nitrophenyl carbonate and 0.5 M in DMAP and mixing for 20 min at rt. Thereafter, the glass support is washed with THF and filtered.
  • the dirnethoxytrityl at the 5' terminus is then removed by washing the support with dichloromethane followed by treatment with 5 ml of 0.2 M dichloroacetic acid in dichloromethane for 5 min at rt.
  • the glass support is next washed with dichloromethane and filtered.
  • Subsequent subunits, prepared as in Example 1 and activated as in Example 2 are added in a like manner and in the following order: G, A , T, A , A ,C, A ,T,T, T,T,T,C, (G protected with the FMOC moiety, A and C protected with the p-nitrophenethoxy carbonylmoiety).
  • Example 21 Preparation of a 19-subunit carbamate-linked polymer targeted against a conserved sequence in the AIDS viral genome. Stepwise assembly of oligorner blocks on a solid support: addition of the first subunit and chain extension.
  • the 5'-amino-2', 5'-dideoxycytidine subunit (0.2 mmol), prepared as in Example 3 (wherein the N4 carries a benzoyl moiety, the 5'-amine carries a p-methoxytrityl moiety, and the 3' oxygen carries a p-nitrophenoxycarbonyl moiety) is dissolved in a minimal volume of dry THF and added to 0.5 g of dried controlled pore glass support carrying a cleavable linker, prepared as in Example 19. Catalyst (either 1 mmol
  • N-methylimidazole or 0.1 mmol of DMAP is introduced and the slurry mixed by rocking or tumbling for 2 h. The slurry is next filtered, and the solid washed thoroughly with THF. The mono-p-methoxy trityl is removed by washing with dichloromethane, followed by treatment with 5 ml of 0.2 M dichloroacetic acid in dichloromethane at rt for 1 min. The support is then washed with dichloromethane and filtered. The 5' amino termini are converted to their free amine form by a brief wash with 3 ml of THF containing 1% by volume of diisopropylethylamine, followed by washing with THF and filtration.
  • Any unreacted amine moieties are capped by adding 2 ml of THF 2 M in p-nitrophenyl acetate and mixing at rt for 20 min. Thereafter, the glass support is washed with THF and filtered.
  • the mono-m ⁇ thoxytrityl at the 5' terminus is removed with dichloroacetic acid, as before.
  • Subunits having base exocyclic ring nitrogen protective groups removable by strong nonnucleophilic bases can also be used for assembling polymers by the procedure above.
  • polymers having these alternative protective groups are generally deprotected by treatment with DBU rather than by ammonium hydroxide.
  • Example 22 Preparation of a 19-subunit amide-linked polymer targeted against a conserved sequence in the AIDS viral genome. Stepwise assembly of oligorner blocks on a solid support: addition of the first subunit and chain extension.
  • a cytosine-containing acyclic-backboned subunit (0.2 mmol), prepared as in Example 11.3 (wherein the N4 of the cytosine carries a 2(P-nitrophenyl)ethoxycarbonyl moiety, the amine of the backbone carries a t-butoxycarbonyl moiety, and the carboxyl is in the form of a p-nitrophenyl ester) is dissolved in a minimal volume of dry THF and added to 0.5 g of dried controlled pore glass support carrying a cleavable linker, prepared as in Example 19. Catalyst (either 1 mmol of
  • N-methylimidazole or 0.1 mmol of DMAP is introduced and the support mixed for 2 h, filtered, and the solid is washed thoroughly with THF.
  • the t-BOC is removed by washing the support with dichloromethane followed by treatment at rt with 5 ml of 20% by volume trifluoroacetic acid in methylene chloride for 30 min.
  • the support is washed with dichloromethane and filtered.
  • the support-bound amino terminus is converted to the free amine form by a brief wash with 3 ml of THF containing 1% by volume of diisopropylethylamine, followed by a THF wash.
  • the activated dimeric subunit (0.1 mmol) having the sequence (amino terrninus)-G-A-(carboxy terminus) prepared as in Example 17 (wherein the N2 of guanine carries an FMOC moiety, the N6 of adenine carries a p-nitrophenethoxycarbonyl moiety, the backbone amino terminus carries a t-BOC moiety, and the carboxy terminus is in the form of a p-nitrophenyl ester) is dissolved in a minimal volume of dry THF and added to the glass support and mixing is carried out for 2 h (no catalyst is added for this or subsequent coupling steps). The support is washed with THF.
  • Any unreacted amine moieties are capped by adding 2 ml of 2 M p-nitrophenyl acetate in THF and mixing at rt for 20 min. Thereafter, the glass support is washed with THF.
  • Example 17 are added in a like manner in the following order: (C-terminus) T-A , A-C, A-T, T-T, T-T, C-A, C-A, A-C (N-terminus).
  • Subunits having exocyclic ring nitrogen protective groups removable by good nucleophiles can also be used for assembling polymers by the above procedures.
  • polymers having these alternative protective groups are generally deprotected by treatment with ammonium hydroxide rather than by DBU.
  • Example 23 Configuring the carbonate-linked polymer of Example 20 for a solution application.
  • the 5' oxygen of the support-bound polymer of Example 20 is succinylated with 5 ml of a THF solution 1 M in succinic anhydride and 0.1 M in DMAP for 10 min at rt, washing with THF, and filtering.
  • the polymer is both released from the support and its recognition moieties are deprotected by treatment with 3 ml of dry DMSO containing 0.3 g of DBU at rt for 12 h.
  • the polymer is freed from DBU by two cycles of precipitation with anhydrous either and resuspension in anhydrous DMSO. Thereafter, the polymer is resuspended in agueous DMSO containing a trace of pH 7.5 buffer to neutralize any residual DBU and purified by anion exchange chromatography.
  • 6-A ⁇ ninocaproic acid is reacted with 2-phenylisopropylphenyl carbonate by the method of
  • the glass support is then washed with THF and filtered the polymer is both released from the support and its recognition moieties deprotected by treatment with 3 ml of dry DMSO containing 0.3 g of DBU at rt for 12 h.
  • the polymer is freed from DBU by two cycles of precipitation with anhydrous ether and resuspension in anhydrous DMSO.
  • the phenylisopropoxycarbonyl group is removed by adding 80% aqueous acetic acid and incubating at rt for 2 h.
  • the deprotected polymer is precipitated with ether, resuspended in aqueous DMSO, and purified by cation exchange chromatography.
  • One gram of cellulose (Type 20, Cat.
  • the activated cellulose (0.5 g) is washed with DMSO, filtered, and 10 ⁇ mol of polymer in 1 ml of DMSO is added followed by 30 ⁇ mol of diisopropylethyl arnine. This mixture is stirred slowly overnight at rt in a closed vessel, filtered, and washed with DMSO. The support is next washed with aqueous pyridine, rinsed with methanol, and dried.
  • Example 25 Configuring the carbamate-linked polymer of Example 21 for a solution phase application.
  • the 5' amino moiety of the support-bound carbamate-linked polymer of Example 21 is succinylated by reacting with 5 ml of a THF 1 M solution in succinic anhydride and 1 M in diisopropylethylamine for 10 min at rt, then washing with THF.
  • the polymer is released from the support by treatment with 3 ml of DMF containing 0.3 g of DBU.
  • the polymer is freed from the DBU by 2 cycles of precipitation with ether and resuspension in DMF.
  • Protective groups are removed from the recognition moieties by precipitating the polymers with ether, resuspending in 1 ml of DMSO, and adding 1 ml of concentrated ammonium hydroxide.
  • the protective group on adenine is a benzoyl moiety, deprotection is carried out in a closed container at 30°C for 12 h.
  • the protective group on adenine is a nitrobenzoyl and the protective group on guanine is an acetyl moiety
  • deprotection is carried out at rt for 6 h. Volatile components are removed under vacuum and the polymer solution neutralized with pH 7.5 buffer. Finally, the polymer, diluted in aqueous DMSO, is purified by anion exchange chromatography.
  • the polymer is released from the support and protective groups on the recognition moieties are removed as in Example 25, except that the final purification is by chromatography on a cation exchange column.
  • the purified polymer is linked to a cellulose support as in Example 20.
  • Example 22 The support-bound amide-linked polymer of Example 22 is succinylated as in Example 24.
  • Example 28 Configuring the amide-linked polymer of Example 22 for a solid phase application.
  • Example 22 The support-bound amide-linked polymer of Example 22 is reacted with activated aminocaproic acid and the phenylisopropoxycarbonyl moiety removed as in Example 24.
  • the polymer is released from the support, its protective groups on the recognition moieties are removed, it is purified, and then linked to a cellulose support essentially as in Example 24.

Abstract

A polymeric composition effective to bind, with a selected binding affinity, to a single-stranded polynucleotide containing a target sequence of bases. The composition includes non-homopolymeric, substantially stereoregular polymer molecules, each composed of a sequence of base complementary recognition moieties adapted to hydrogen bond to corresponding, in-sequence bases in such target sequence, under selected binding conditions, and a substantially stereoregular, predominantly uncharged backbone supporting the recognition moieties at positions which allow hydrogen bonding between the recognition moieties and the corresponding in-sequence bases in the target sequence. The composition is useful in a diagnostic assay system for determination of a polynucleotide containing the target sequence; in research applications involving binding to specific target sequences in solution for the purpose of selectively altering target expression, including replication, transcription, and translation; and in therapeutic uses based on inactivation of single stranded polynucleotides in vivo.

Description

STEREOREGULflR POLYNUCLEOTIDE-BINDING POLYMERS
1. Field of the Invention
The present invention relates to base-specific polynucleotide-binding polymers
2. Background of the Invention
Compounds capable of recognizing and binding specifically to a target base sequence in a polynucleotide have many potentially valuable applications. For example, in therapeutic uses, the compounds could act to bind to and inactivate viral genomes, or other single-stranded polynucleotide species characteristic of an infecting microorganism. In diagnostic applications, the compounds could be used to "hybridize" with analyte single-stranded polynucleotides, to detect the presence of viral or bacterial-specific nucleic acid species.
The earliest attempts to devise an agent which is capable of binding specifically to a base-specific target region of a polynucleotide were based on the concept of (Belikov, Zarotova, Grinev) derivatizing a target-specific polynucleotide with an alkylating agent, (Summerton and Bartlett (1978), J. Molec. Biol, 122: 145) binding the derivatixed copound to the target single-strand RNA or DNA. Alkylation of the target strand would then bind the compound irreversible to the target. There are two major difficulties with this approach. First, the negatively charged backbone of the derivatized polynucleotide seriously restricts its transport into living cells. Second, the phosphodiester linkages in the agents are susceptible to rapid cleavage by cellular nucleases -- particularly when the gene inactivating agents are taken into cells via endocytosis.
More recently, recombinant DMA methods have allowed for the design of plasmids which carry a anti-sense gene whose RNA transcript can recognize and bind to a particular target single-strand polynucleotide. Introduction of such a plasmid into cells results in continuing production of anti-sense mRNA which appears to pair with its complementary target mRNA and thereby block its translation (Izant and Weintraub (1984), Cell, 36:1007; Izant and Weintraub (1985), Science, 229 :345). A particular merit of this approach is that successful introduction of even a single such plasmid could potentially effect continued inactivation of the targeted gene activity and/or permanent resistance to the pathogen containing the targeted genetic sequence. The approach is severely limited, however, by the problems of transforming a large number of tissue or organ cells with the plasmid construct.
Attempts to design a polynucleotide binding agent which is both readily taken up by cells and stable against intracellular degradation have focused on polynucleotide analogs which have uncharged or substantially uncharged backbones. One of the earliest reports of such uncharged comppounds involved a number of polynucleotide analogs in which the normal sugar-phosphate backbone of nucleic acids was replaced by a polyvinyl backbone. These nucleic acid analogs were shown to have normal Watson/Crick pairing specificities with complementary polynucleotides -- but with substantially reduced Tm values (Pitha and Pitha (1970), Biochem. Biophys. Acta, 204:39). Subsequently, uncharged and substantially uncharged heteropolymeric polynucleotide analogs more nearly isostructural with nucleic acids have been prepared for the purpose of in vivo inactivation of targeted single-stranded genetic sequences. These substances include both aryl nitrogen mustard derivatized (Karpova et al (1980), FEBS Letters, 122:21) and underivatized (Richard Tullis, Personal. Communication, Molecular Biosystems, Inc., San Diego) oligonucleotides having their phosphates in the uncharged triester state. A novel class of polynucleotide analogs which has recently been reported contain predominantly uncharged, atactic methylephosphonate backbone linkages, or homochiral methylphosphonate linkages alternating with charged, achiral phosphodiester linkages (Miller, Yano, Yano, Caroll, Jayaraman, and Ts'o (1979), Biochem., 18:5134; Miller, Dreon, Pulford, and Parland (1980), J. Biol. Chem., 255:9659; Murakami, Blake, and Miller (1985), Biochem., 24:4041; Blake, Murakami, Spitz, Glave, Reddy, Ts'o, and Miller (1985), 24:6132; Miller, Agris, Aurelian, Blake, Murakami, Reddy, Spitz, and Ts'o (1985), Biochimie, 67:769).
The above-described uncharged or substantially uncharged oligonucleotide analogs have been reported to enter living cells and pair with complementary genetic sequences therein. In particular, it has been found that optimal uptake by the cells is achieved if the compounds are designed with one or a few ionic groups per molecule, to enhance solubility in aqueous medium. If the charge density on the backbone is increased significantly, however, problems of poor transport across the cell membrane are encountered. Also, greater stability to nucleases is achieved if phosphodiester linkages are avoided.
The above uncharged and substantially uncharged polynucleotide analogs all have chiral backbone linkages, i.e., linkages in which two possible stereoisomeric forms are allowed. As a consequence, the backbone of the polymer produced by linking subunits has a random sequence of left- and right- handed forms. This type of backbone structure, which is also referred to as an atactic backbone, is characterized by a broad range of analog/target binding constants. The mean binding constant can be significantly reduced with respect to the binding constant between normal complementary polynucleotides.
For the phosphotriester and methylphosphonatelinked nucleic acid analogs this binding constant reduction and broadening presumably derives from one linkage isomer favoring Watson/Crick pairing of proximal bases and the other linkage isomer inhibiting Watson/Crick pairing of proximal bases. This linkage chirality effect on base-pairing has been well established by studies involving two separated isomeric forms of a methylphosphonate-linked dideoxyribonucleoside. Each of these dimers was mixed with its complementary polyribonucleotide. One isomer was found to bind with a Tm of 19.8°C while the other isomer had a corresponding Tm value of only 15.4°C (Miller, Dreon, Pulford, and Parland (1980), J. Biol, Chem, 255:9659). These studies were further extended to examine Tm binding values in homoisomeric decamers formed by linking the isomeric methylphosphonate dimers with normal phosphodiester linkages. The decamer formed from dimers having the preferred isomeric methylphosphonate linkages paired with its complementary genetic sequence with a Tm of 33°C. By contrast, the corresponding decamer containing the other isomeric form of methylphosphonate linkages failed to pair with its complementary genetic sequence even at 0°C (Miller, Dreon, Pulford, and Parland (1980), J. Biol, Chem, 255:9659).
On theoretical grounds there is reason to believe that for a sequence-specific polynucleotidebinding agent to be of therapeutic and/or diagnostic value, it should pair with its complementary target sequence with a substantially uniform binding constant. The rationale here is that if a given preparation contains multiple molecular species, each with its own separate target binding constant, then those species having lower binding constants will contribute little to the target binding activity while some of those having significantly higher binding constants also form moderately stable mispaired complexes with nontarget sequences. In a therapeutic context any significant binding to nontarget sequences has the potential of generating toxic side effects, while in a diagnostic context such binding is expected to lead to high backgrounds and/or false positives.
In principal, using currently available technologies it should be possible to prepare the foregoing charged or partially charged and chirally linked nucleic acid analogs having substantially uniform target binding constants. One method for accomplishing this would involve binding the the polymer solution, which contains polymer species with a wider range of Tm values for a given target sequence, to an affinity column having the target sequence in an immobilized form. The analog is then eluted with with a gradient of an eluant, such as formamide, which will release increasingly more tightly bound analog at higher eluant concentrations, and/or by eluting while slowly increasing the temperature. A narrow cut of the eluant should contain primarily those molecules having a substantially uniform target binding constant. Achieving greater binding homogeneity with this method obviously means reducing the final yield of usable polymer substqntially. Alternatively, it is possible that uncharged oligonucleotide analogs having tactic linkages (i.e., all molecules having the same sequence of chiral centers) could be prepared by means of a geometric assembly procedure in which one of the two stereoisomers formed at each subunit addition step is retained, and the other discarded. The limitation of this procedure, of course, is that at least half of the material is discarded at each subunit addition step, so that the yield can be at most 1/2 of the starting material, where n is the number of rounds of couplings required in the synthesis.
Thus, although it may be possible to prepare uncharged polynucleotide analogs having chiral intersubunit linkages which pair with their complementary target sequences with a substantially uniform binding constant, nevertheless, the synthesis and purification of such chirally linked polymers would be time-consuming and yields would necessarily be poor.
3. Summary of the Invention
It is therefore one general object of the invention to provide a base-specific polynucleotide-binding composition which substantially overcomes problems and limitations associated with prior art polynucleotide-binding agents. Related objects of the invention include:
(a) Providing such a composition whose polymer molecule components all have substantially the same binding affinity with respect to a target sequence in a single-stranded polynucleotide;
(b) Providing such a composition which can be designed for targeting against virtually any nucleotide target sequence within a length range of between about 5-30 base pairs, and which can be prepared in relatively high yield;
(c) Providing such a binding composition having a desired binding affinity for a selected target sequence; and
(d) Providing such a binding compound which is suitable for a variety of diagnostic and therapeutic applications which take advatage of the of the stereoregular, uncharged or substantially uncharged nature of the backbone linkages in the compound.
The invention includes a polymeric composition designed to bind, with a selected binding affinity, to a single-stranded polynucleotide containing a target sequence of bases. The composition includes non-homopolymeric, substantially stereoregular polymer molecules of the form:
R1 R2 R3 Rn
B ~ B ~ B ~ . .. B, where:
(a) RI-Rn are recognition moieties selected from purine, purine-like, pyrimidine, and pyrimidine like heterocycles effective to bind by Watson/Crick pairing to corresponding, in-sequence bases in the target sequence;
(b) n is such that the total number of Watson/Crick hydrogen bonds formed between a polymer molecule and target sequence is at least about 15;
(c) B ~ 8 are backbone moieties joined by chemically stable, substantially uncharged, predominantly achiral linkages;
(d) the unit length of backbone moieties is 5-7 atoms if the backbone moieties have a cyclic structure, and 4-6 atoms if the backbone moieties have an acyclic structure; and
(e) the backbone moieties support the recognition moieties at positions which allow Watson/Crick base pairing between the recognition moieties and the corresponding, in-sequence bases of the target sequences.
In a preferred embodiment for use in therapeutic applications, the polymeric molecules, have a small number of charges, to increase compound solubility in aqueous medium. The charges may be attached at end regions of the molecules or at achiral charged backbone linkages.
The polymer molecules are constructed, according to the method of synthesis of the invention, by first selecting a sequence of bases in the target polynucleotide. The sequence is typically between about 5-32 bases, and preferably between about 8-20 bases, corresponding to the number of subunits in the final binding polymer. The binding affinity of the binding polymer for the target polynucleotide can be increased by increasing the number of target-sequence bases (and correspondingly, the number of subunits in the binding polymer). Polymer subunits of the form B-Ri and/or B-Rj are then coupled in a defined sequence corresponding to the sequence of bases in the target-specific sequence. Here Ri and Rj are recognition moities, as defined above, which are capable of forming Watson/Crick base pairing with corresponding polynucleotide bases through either two or three hydrogen bonds, respectively. The binding affinity or the Tm of the binding polymer for the target polynucleotide can be selectively varied according to the ratio of Ri to Rj recognition moieties used in forming the polymer, with a greater percentage of Ri recognition moieties producing a lower binding affinity for the target polynucleotide.
The polymers are useful in a diagnostic system for detecting an analyte polynucleotide having a defined target base sequence. The system includes a support reagent composed of the polymer molecules of the invention linked to a solid support. In one embodiment of the invention, the polymer binding molecules are predominantly uncharged. In another embodiment, the backbone moieties are joined by achiral charged linkages, such as phosphodiester linkages, alternating with one or more uncharged achiral linkages.
In practicing the assay method, a polynucleotide analyte is added to the diagnostic reagent under conditions in which the analyte is in a single-strand form and which allow sequence-specific pairing between the analyte and the reagent polymers. After the analyte/polymer annealing reaction, the reagent is washed to remove unbound test material. The reagent and bound analyte are then reacted with a oligocationic reporter which is designed to bind by charge attraction to the charged, phosphodiester linkages of the bound analyte, but not to the uncharged or substantially uncharged polymer molecules. One feature of the system is that the ratio of bound reporter to bound analyte polynucleotides (the number of reporter molecules bound per analyte molecule) can be 102-104, giving a highly sensitive assay.
In therapeutic applications, the polymer composition is designed to inhibit expression of a selected target single-stranded polynucleotide, such as an RNA virus. Here the polymer is preferably constructed to bind spsecifically to the target polynucleotide, with the minimum binding needed to produce the desired inhibition of target expression, to minimize non-specific binding effects.
These and other objects and features of the present invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying figures.
Brief Description of the Figures
Figure 1 shows preferred purine and pyrimidine structures used in forming polymer molecules of the invention;
Figure 2 shows preferred purine-like and pyrimidine-like recognition moieties used in forming the polymer molecules;
Figure 3 shows preferred cyclic backbone moities used in forming the polymer molecules;
Figure 4 shows preferred acyclic backbone moieties used in forming the polymer molecules;
Figures 5A and 5B illustrate two preferred subunit assembly schemes for coupling N1---N2 type subunit backbones;
Figure 6 shows the backbone structures of subunit dimers A-A through D-D formed in accordance with the methods illustrated in Figures 5A and 5B, and two cyclic backbone structures formed in accordance with the methods illustrated in Figures 7A and 7B;
Figures 7A-7C illustrate three preferred subunit assembly schemes for coupling N1---E type subunit backbones; and
Figure 8 shows preferred acyclic backbone structures of subunit dimers formed in accordance with the methods illustrated in Figures 7A-7C;
Detailed Description of the Invention
The polymeric composition of the invention is designed to bind, with a selected binding affinity, to a polynucleotide containing a target sequence of bases. The composition is composed of non-homopolymeric, substantially stereoregular molecules or species of the form:
R1 R2 R3 Rn
B ~ B ~ B ~ . . . B,
where B-Ri are a series of base-specific subunits containing a backbone moiety B and a recognition moiety
Ri which is selected to bind specifically by
Watson/Crick base pairing to a corresponding, in-sequence base in the target sequence, and the subunits are joined through their backbone moieties predominantly achiral, substantially uncharged bonds.
The design and selection of suitable subunits for use in constructing the polymer species will be be described in
Section I below. Methods for coupling subunits through achiral linkages are described in Section II, with subunit-protective group strategies involved in subunit synthesis being discussed in Section II.
The polymer species are synthesized by sequential subunit coupling, to form polymer molecules of a selected length and sequence. As will be described in Section IV, the length of the target sequence and polymer sequence is selected to achieve a desired binding specificity. The binding affinity of the polymer for the target can be selectively varied by several strategies discussed in Section IV, including the choice of recognition moieties which are capable of forming either three (for greater binding affinity) or two (for lesser affinity) base-paired hydrogen bonds with the corresponding target base. The resulting composition contains polymer species or molecules, all having substantially the same sequence of subunits and substantially the same sequence of intersubunit linkage types. In functional terms, all of the polymer species have substantially the same binding affinity for the target polynucleotide. Methods for assembling the polymer, once a desired subunit sequence is selected, are given in Section IV.
The polymer composition is useful in a novel solid-phase diagnostic system which is described in Section IV. This system is based on the binding of analyte polynucleotide molecules to support-bound polymer molecules and subsequent elctrostatic attachment of multiple polycationic reporter molecules to the polynucleotide, to give an amplified reporter signal for each bound analyte molecule. The polymer is also useful in a variety of solution applications, including therapeutic applications, which are also considered in Section IV. I. Subunit Structure
A. The Recognition Moiety
The design of the subunits B-Ri which are suitable for use in forming the polymer species of the invention involves a number of structural and/or stereochemical criteria which must be met both by the backbone and recognition moieties and by the linkage between the two. The design requirement of the recognition moiety will be considered first.
The recognition moiety of each subunit must provide two or three hydrogen-bonding groups held in an unambiguous configuration adapted for hydrogen bonding to two or three of the Watson/Crick hydrogen bonding sites on a specified in-sequence base of the target genetic sequence. To avoid undesired mispairing between recognition moiety and its corresponding target base, under the conditions of use, (1) the tautomeric state of the recognition moiety should be relatively stable, and (2) the recognition moiety should have a structure which provides a relatively rigid arrangement of the hydrogen-bonding groups. Such rigidity is best afforded by a ring structure having the polar hydrogen-bonding groups either forming part of the ring, or directly attached to the ring.
The preferred recognition moiety structures include purine, purine-like, pyrimidine, and pyrimidine-like structures which are designed to form Watson/Crick base pairing, through either two or three hydrogen bonds, with selected polynucleotide bases. The group of subunits used in polymer synthesis includes at least two recognition moieties which are base-specific for different polynucleotide bases, and preferably one or more recognition moieties for each of the four bases in a natural DNA or RNA polynucleotide. Also, as will be seen below, it is desirable to provide one group of recognition moieties, Ri, which are capable of base-pairing with nucleotide bases through two hydrogen bonds, and a second group, Rj which are capable of binding with the same bases through three hydrogen bonds. Figure 1 shows exemplary purine and pyrimidine type recognition moieties. The purine structures 1 and
2 are designed to bind to thymine or uracil bases, structures 3-6, to guanine bases; structures 7-9, to cytosine bases, and structures 10-12, to adenine bases.
Structures 1, 4, 5, 6, 8, and 10-12 are Ri type moieties adapted to bind to corresponding in-sequence bases through two hydrogen bonds, and the remaining structures are Rj type moieties which form three hydrogen bonds on base pairing. As will be seen below, purine and pyrimidine nucleosides are useful in synthesizing a variety of other subunits which are suitable for use in polymer synthesis. These subunits, modified if necessary with amine protective groups, can be obtained from commercial sources, or prepared according to known methods, such as those described or referenced in Example 1.
A number of purine-like or pyrimidine-like structures in which the recognition moiety is joined to the backbone through an annular carbon are shown in
Figure 2. These structures have base pairing specificities and hydrogen bonding properties like those of the analogous structures shown in Figure. Although the binding properties of a polymer containing a carbon-linked recognition moiety may not be significantly different from that of a polymer whose recognition moities are nitrogen-linked, as in Figure 1, subunits incorporating the carbon-linked moieties generally require a greater synthetic effort than do subunits containing preformed purine and pyrimidines. However, for some subunit syntheses, such as the synthesis of the achiral acyclic backbone subunits described in Example 10, the carbon-linked recognition moieties provide a useful starting material.
B . The Backbone Moiety
The backbone moiety of each subunit has the general form N1---E, or N1---N2, where N1 and
N2 are nucleophilic groups and E is an electrophilic group. Based on ease of subunit coupling and required stability of the resultant intersubunit linkage, as discussed below, the preferred nucleophilic groups are amino, hydroxyl and hydrazino; and the preferred electrophilic groups, and/or electrophilic linking agents, are derivatives of carbonic, thiocarbonic, carboxylic, and sulfonic acids.
Backbone moieties can have either a cyclic or an acyclic structure. While the total number of possible backbone moiety structures is very large, nevertheless, only a rather limited number of structures are of actual practical value due to a number of factors. The initial procedure by which promising backbone moieties were selected was as follows.
As a first conditions, only those cyclic backbone moieties were considered which consisted of, or could be readily derived from, deoxyribose or ribose. This limitation is a practical one and reflects the difficulty and corresponding greater expense of de novo synthesizing ring structures having multiple chiral centers. In general prospective backbone moieties and intersubunit linkages expected to be suitable for polymers were selected on the basis of one or more of the following factors: feasibility of synthesis based on known reactions; expected ease of synthesis from available starting materials; simplicity of structure; and expected stability of the final backbone.
Initial screening of promising backbone moieties and intersubunit linkages was performed as follows. Space-filling CPK molecular models of duplex DNA and RNA were constructed according to parameters determined by x-ray diffraction of oligodeoxyribo nucleotides in the B-form and oligonucleotides in the A-form. In each of these constructed duplexes one of the two sugar-phosphate backbones was removed. Next, each prospective backbone was attached, if possible, to the sites on the bases form which the original sugar-phosphate backbone had been removed. Each resulting polynucleotide/polymer duplex was then examined for coplanarity of the Watson/Crick base-pairs, torsional and angle strain in the prospective polymer backbone, degree of distortion imposed on the nucleic acid strand, and interstrand and intrastrand nonbonded interactions. Special attention was paid to whether or not each amide-containing backbone could readily adopt a conformation wherein its amide moieties were planar. This is important because of the substantial energy cost required to force an amide into a nonplanar conformation.
These initial studies verified that the required unit backbone length (i.e., the N1---E spacing in an N1---E or activated N1---N2-E subunit) is 5-7 atoms for backbone moieties constituting or derived from deoxyribose or ribose (cyclic backbone structures), with a 6-atom length being optimal.
Subunit structure judged acceptable in the above modeling studies were then assessed for feasibility of synthesis (on the basis of key synthetic reactions reported in the literature, or reactions carried out on model compounds, and/or via actual synthesis of the subunits) and stability of the assembled polymer backbone (preliminary stability studies were generally carried out with suitably linked model compounds or subunit dimers). These types of studies were used to further restrict the number of candidate backbone structures. From this restricted candidate pool the cyclic backbone structures A-G shown in Figure 3 were, ultimately selected as preferred structures.
In this figure, subunits A-D containing N1----N2 type cyclic backbone moieties include:
2'-deoxyribonucleosides (structure A); 2'-deoxyribonucleosides substituted at the 5' and 3' positions with an amino group (structures B and C, respectively); and morpholino derivatives of ribonucleosides (structure D). Subunits containing
N1---E-type backbone moieties include
2'-deoxyribonucleosides substituted at their 5' positions with one and two-carbon acids (structures E and F, respectively); and N-aminomorpholino derivatives of ribonucleosides substituted at their 5' positions with carboxylic acid (structure G) or sulfonic acid (structure H).
The same molecular modeling approach applied to acyclic (unbranched chain) backbone moieties showed that unit backbone lengths ranging from 4-6 atoms are acceptable, in terms of polymer conformation in base-pair hydrogen bonding to single-stranded polynucleotide, with a 5-atom backbone length being optimal. This is in contrast to cyclic backbones, where a 6-atom unit backbone length is optimal. Further, the modeling work showed that the annular recognition moiety cannot be directly attached to the linear backbone moiety without severely inhibiting of coplanarity of the complementary bases and introducing undesirable interactions between the recognition moiety and the backbone the minimum binding strain occurred when the recognition moiety was linked to the backbone moiety by a one-atom spacer. Two-atom, but not three-atom, spacers may be tolerated, although increased degrees of freedom between the recognition moieties and the linear backbone may lead to mispairing with target bases with two-atom spacers.
The acylic backbone moiety structures I-N shown in Figure 4, all of which contain a one-atom methylene spacer between the backbone and recognition moieties, gave a good modeling prediction for favorable base-pairing to a cornplementray polynucleotide. Analogous structures containing a two-carbon spacer showed acceptable, but less favorable, binding conformation. The structures shown in the figure, and the analogous two-carbon spacer structures, are preferred acylcic backbone moieties because of their general ease of synthesis. It should be mentioned, however, that a number of other backbone moieties are also entirely feasible and suitable -- although generally not as easy and/or inexpensive to synthesize.
C. Backbone/Recognition Moiety Linkage
As indicated above, the linkage or spacer connecting the backbone and recognition moieties in the polymer subunit must meet certain design criteria which are effective to position the recognition moieties for base-pair binding to the polynucleotide bases. In the case of the cyclic backbone moieties shown in Figure 3, modeling studies indicate that the most favorable binding conformation in the polymer occurs when the recognition moiety is attached directly to the 1' carbon of the ribose or ribose-like structures, and to the analogous 1' position of the morpholino structures. That is, the moiety is attached at normal point of attachment and with the normal stereoisomeric configuration of purine or pyrimidine bases to the ribose or deoxyribose groups of nucleosides. For the acyclic structures, one-and two-carbon atom spacers are required for placing the recognition moieties at favorable binding positions, with one-atom spacers being preferred, as discussed above.
In order to achieve a stereoregular polymer configuration required for uniform binding affinity of the polymer molecules to a polynucleotide target, it is also necessary that all of the backbone/recognition moiety linkages be either of define chirality or achiral. The linkages are considered to be of defined chirality, for purposes of definition herein, if each of the linkages in any given subunit position in all of the polymer molecules has the same stereoisomeric configuration, or chirality. That is, although the subunits in different sequence positions may have different internal stereoisomer configurations (including achirality at specific sequence positions), all of the linkages at a given sequence position among the polymer molecules have the same stereoisomeric configuration. Usually defined chirality is most easily achieved by using the same stereoisomeric configuration in all of the subunits.
For the cyclic backbone moieties, the most favorable binding occurs in nucleoside analogs having the natural D-steriosomeric configuration shown in Figure 3. Subunits of this type are also readily synthesized, as will be discussed below, using natural D-nucleoside starting material. With reference to Figure 4, it is seen that only structures I and L have chiral linkages between the backbone and recognition moieties. These subunits are readily synthesized in homochiral form by using homochiral starting material , as will be described. For all of the other acyclic structures shown in Figure 4, the one-atom linkages are attached to backbone nitrogen atoms, are are therfore achiral. Also, of course, the methylene spacers are themselves achiral.
II. Protective Group Strategies
Because of the rather high reactivity of the compounds used for activating and/or coupling the subunits, it is generally desirable, and often necessary, to protect the exocyclic ring nitrogens of the recognition moieties. Selection of these protective groups is primarily determined by the type of intersubunit linkage to be used in the polymer assembled from these subunits, and secondarily determined by the relative reactivity of the nitrogen to be protected.
Base-protected nucleosides are also useful starting reagents in a number of the subunit synthesis reactions to be described below. Methods for base protecting a number of the more common ribo- and deoxynucleosides from Example I are illustrated in Example 2. The methods detailed in the example are generally applicable for forming nucleosides with amine-protective groups.
When the intersubunit linkage to be used is relatively stable to nucleophiles, and particularly to ammonium hydroxide, then the standard base-protective groups used for nucleic acid chemistry are suitable. Such nucleophile-insensitive intersubunit linkages include carbamate, amide, and sulfonamide linkages. The corresponding nucleophile-sensitive protective groups for the recognition moieties include: benzoyl for the N4 of C; benzoyl or p-nitrobenzoyl for the N6 of A; acetyl or isobutyryl for the N2 of G; and N2,N6-bisisobutyryl for 2,6-diaminopurine residues. Removal of these groups after completion of polymer assembly is effected by treatment with ammonium hydroxide.
In contrast, when the intersubunit linkage to be used is sensitive to nucleophiles, such as ammonium hydroxide, suitable protective groups are those which can be removed by strong non-nucleophilic bases -- via a β elimination mechanism. Such nucleophile-sensitive intersubunit linkages include: carbonate; ester; and, to a lesser extent, triocarbamate linkages. Suitable protective groups for the recognition moieties removable via β elimination include: 2-(4-nitrophenyl)ethoxy carbonyl or 2-(phenyl sulfonyl) ethoxycarbonyl for both the N4 of C and the N6 of A; and the 9-fluorenyl methoxycarbonyl for the N2 of G and the N2 and N6 of 2,6-diaminopurine residues. Removal of these groups after completion of polymer assembly is effected by treatment with the strong nonucleophilic base 1,8-diazabicyclo[5.4.0]uπdec-7-ene (DBU), under stringently anhydrous conditions.
In regard to temporary protection of a nucleophile of the backbone moiety ( generally N 1 in the abov e s tructures), in the general polymer assembly strategy employs selected backbone-protective groups which are readily cleared by mild acid. One primary criterion for selection of protective groups is that they be adequately stable, but not so stable that conditions required for their removal would damage the growing polymer. A principal problem in the case of polymers assembled from cyclic backbone moieties is the particular acid sensitivity of the glycosidic bond linking protected purine residues to the C1 of their backbone moieties. A secondary criterion for selection of the backbone protective group is that it be easily introduced. Based on the above, the following backbone-protecting groups are preferred: for primary hydroxyls, the di(p-methoxy) trityl group; for primary amines, p-methoxytrityl; and for a secondary amine (as in morphilino-type backbone moieties), the phenylisopropoxycarbonyl group. These protective groups can be readily removed by treatment with 0.2 M dichloroacetic acid in dichloromethane.
Ill. Subunit Synthesis
A . Cyclic Backbone Moieties
Subunits having a deoxydeoxynucleoside subunit structure (structure A in Figure 3) can be obtained from commercial sources or prepared via literature methods, as described in Example I. The subunits include the following ribosides and deoxyribosides, which are identified according to the struture numbers of the recognition moieties given in Figure 1: adenosine and deoxyadenosine (structure 1); 2,6-diaminopurine riboside and deoxyriboside (structure 2); cytodine and deoxycytodine (structure 3); 4-methoxy-2-pyrimidinone deoxyriboside (structure 4); 2-hydroxy-5-methyl pyrimidine deoxiriboside (structure 5); 2-hydroxypyrimidine riboside (structure 6); guanosine and deoxyguanosine (structure 7); inosine and deoxyinosine (structure 8); thioguanosine and deoxythioguanosine (structure 9); uradine and deoxyuridine (structure 10); thymidine and 5-methyluridine (structure 11;) and 5-halouridines and 5-halodeoxyuridines (structure 12).
The 5'-amino-2',5'-dideoxyribonucleosides (structure B in Figure 3) are prepared according to methods detailed in Example 3. Briefly, the selected deoxyribonucleosides, base-protected if necessary, are reacted with triphenyl phosphine, carbon tetrabromide, and lithium azide to form the corresponding 5 '-azidonucleoside, which is then reduced by hydrogenation in the presence of a palladium on carbon catylyst. The nucleosides may be obtained as in Example 1, and base-protected as in Example 2. The stereochemistry of the reactant nucleosides is preserved in forming the 5' amino nucleoside analogs.
An alternative reduction method is used in reducing the azide group of 5'-azido-5-bromo uridine, as described in Example 3.2. Here non-catalytic hydrogenation is needed to prevent removal of the ring bromine atom. In forming the 5'-amine guanosine compound, the azide is placed on the 5' position by first tosylating the 5' hydroxyl group, then displacing with azide, as detailed in Example 3.2.
The 3'-amino-2',3'-dideoxyribosnucleosides (structure C in Figure 3) are prepared according to methods detailed in Example 4. Briefly, thymidine which is protected at its 5' hydroxyl is tosylated at its 3' hydroxyl, and this is followed by an intramolecular displacement involving the 2 oxy base substituent. The resulting ring is now opened by treatment with azide, yielding a 3' azido analog of the correct stereoisomeric form. This analog can be be converted to a selected-base analog by reacting the azide compound with a selected purine or pyrimidine base. The latter may be base protected, if necessary, for subsequent subunit coupling reactions, as described below. The thymidine or other azide analog is then reduced to produce the desired 3' amine nucleoside. The stereochemistry is of the thymidine starting material is preserved in the synthesis.
Synthesis of the morpholino-type subunit derivatives represented by structure D in Figure 3 are detailed in Example 5 for a variety of different recognition moieties. Briefly, a selected nucleoside, base-protected if necessary, is dissolved in an ammonium salt, such as ammonium biborate, and then reacted with sodium periodate to form transient 2', 3' dialdehydes, which then close upon an ammonium ion to form morpholino ring having 2' and 4' hydroxyls. The compound is then treated with sodium cyanoborohydride to remove the ring hydroxyls. The ring nitrogen is preferably protected as a 2-phenylisopropylcarbamate for subsequent subunit coupling. The sterochemistry of the nucleoside starting material is retained.
Deoxyribose subunit structures having a 5' acetic acid group (structure F in Figure 3) can be prepared according the general synthetic scheme described in Example 6, which details the synthesis of the 5' acetic acid compounds represented by structure F. Here a selected deoxyribonucleoside which is protected at the 3' hydroxyl is converted to the 5' aldehyde, and subsequently treated with a 2-carbon Wittig reagent to give the unsaturated acetic acid derivative. Reduction of the side-chain olefin gives the desired 5' acetic acid compound. The reaction preserves the sterochemistry of the starting nucleoside material The analogous 5' formate compound (structure E in Figure 3) is formed by treating the above 5' nucleoside aldehyde with a one-carbon Wittig reagent, and hydroslysing the resulting enol to form form the corresponding formaldehyde derivative, which is oxidized to the desired acid. The reaction preserves the stereochemistry of the starting nucleoside material.
B. Subunit Synthesis-Acyclic Backbones
The 5-atorn chain amino acid subunit represented by Structure I in Figure 4 is prepared according to the general procedures outlined in Examples 7-9. Briefly, an (S)-5 carboxy pyrrolidone was converted to the corresponding stereochernically pure
(S)-5-tosylmethyl-2-pyrrolidone by known methods, and then reacted with a selected purine or pyrimidine, to form the corresponding (S)-5-methylρurine or pyrimidine pyrrolidone. The displacement reaction retains the original stereospecificity, so that the subunits which are formed are homoisomeric at the site of attachment of the recognition moiety to the backbone carbon.
The purine used in subunit synthesis is preferably contains an electron-withdrawing group at the 6 position, to reduce the reactivity of the 1 and 3 ring nitrogens, i.e., to minimize pyrrolidone coupling at a ring nitrogen. The pyrrolidone-derivized purine or pyrimidine may then be converted to the amine derivative, and base protected, if necessary, prior to the ring opening step which will be described below. Example 7.1 and 7.2 detail methods for forming the cytosine pyrrolidone and its base-protected derivative. The adenosine pyrrolidone formed in Examples 7.3-7.5 employs 6-chloropurine as the starting material, and the corresponding pyrrolidone is converted to the adenosine derivative through azide formation as described. The adenosine is base-protected, as in Example 7.6, prior to ring opening. A similar approach is used to generate base protected guanine pyrrolidone, starting from 2-amino-6 chloropurine, as described in Examples 7.7-7.9. Similar methods are used in synthesizing 2,6-diaminopurine pyrrolidone, inosine pyrrolidone, and 2-hydroxypyrirnidine pyrrolidone, also as detailed in Example 7.
The pyrrolidone is then treated via a series of reactions which cleave the pyrrolidone ring to form an amino acid with a t-BOC protected amine. Examples 8.1-8.4 describe the synthesis of such t-BOC amino acids having one of a number of selected recognition moieties. In a final step (Example 5), the t-BOC protective group may be removed to form the corresponding amino acid represented by structure I in Figure 4. The amino acid may be used directly for subunit coupling, according to reactions given below. Alternatively, the t-BOC protected compound may be activated at its acid group for use in subunit coupling reactions.
The strategy for forming the subunit represented by structure J in Figure 4 is outlined in Examples 10.1 and 10.2, for forming the cytodine and uridine analog subunits, respectively. Briefly, in forming a pyrimidine type subunit, a 6-member heterocycle, such as a pyrimidine or nicotinate, is modified to contain a reactive methylene on the carbon ring position at which attachment to the backbone is desired. As detailed in Example 10.1, the cytosine analog is formed by reacting 5-amino-5-bromopyrimidine to form the corresponding 5-position carboxaldehyde. In Example 12, the uridine analog is formed by converting 6-hydroxynicotinic esters to the corresponding benzylic alcohols. This compound is converted to the aldehyde by reaction in the presence of manganese dioxide.
The aldehyde compound is then reacted with the desired amino acids, such as 4-aminobutyric acid, with reaction at the amine group producing an unstable imine which is reduced to form the stable amine subunit. The secondary amine can be protected, for subunit coupling involving activation of the acid group, by a tBOC group as described in Example 10.1. The base attachment to the backbone at a backbone nitrogen, through a methylene spacer, insures that the subunits lack chiral centers and so cannot be stereoregular.
To form the subunit structures shown at K in Figure 4, the C-carbon backbone analog of the above compound is prepared, using 3-aminoproprionic acid, and this compound is further treated with nitrous oxide to form the corresponding N-nitroso compound, which is subsequently reduced with H2IPd in the presence of iron salts to give the hydrazino acid subunit.
II. Backbone Coupling Reactions
The coupling reactions used in forming the polymer molecules of the invention are stepwise reactions which join one selected subunit or subunit sequence to another selected subunit or subunit sequence. For purposes of the present discussion, the coupling reactions will be described generally with respect to coupling a single subunit B-R1 having a selected recognition moiety R1 to another single subunit B-R2 having the same or a different selected recognition moiety R2, to form a dimer of the form R1 R2
B B where R1 and R2 have the specific sequence shown.
A. Subunit Coupling: N1----N2 Backbone
Configurations
General methods for coupling subunits having an
N1--------N2 backbone configuration are illustrated in Figures 5A and 5B. As will be recalled from Section
I above, N1 and N2 are nucleophilic backbone groups, such as hydroxyl and amine groups, which can be activated with an electrophile E, to form an activatedN1-E or N2-E backbone group which can then react with a second N1----N2 type backbone moiety to form an N1----N2-E-N1----N2 backbone-linked dimer.
The subunit backbones of this types which have been described specifically above are the cyclic backbone structures A-D in Figure 3. In structures A and B, the activated N2 nucleophile is the 3' hydroxyl group in structure C is the 5' hydroxyl and in structure D, the
6' position hydroxyl corresponding to the 5' position hydroxyl in structure C.
In a preferred coupling method, which is illustrated in Figure 5A, a subunit having a selected recognition moiety R1 is activated with an electrophile E. The star at the recognition moiety indicates any required base protection. As seen in the figure, the selected subunit is protected at its N1 nucleophile, to ensure that (a) only the N2 nucleophile is activated and (b) the activated subunit cannot self-polymerize. In structures A, C, and D in
Figure 3, in which the backbone protection group is on the 5' hydroxyl, the protective group is preferably an acid-labile group, such as dimethoxytrityl (DMTO) .
The activating reagent shown in Figure 5A has the general form:
X Y--C--Y, where X is oxygen or sulfur, and C is an active electrophile capable of reacting with a nucleophile, such as a hydroxyl oxygen or amine nitrogen, with displacement of Y, to form the activated subunit:
Activating agents, such as bis-p-nitrophenyl carbonate, which give the carbonyl activated subunit (X=O), are used in forming carbonate and carbamate subunit linkages. Similarly, activating agent, such as thiocarbonyl-di-(1,2,4-triazole) (X=S) are used in forming thiocarbamate linkages.
Subunit activation reactions involving carbonyl activated subunits are detailed in Example 11 for the 5'-protected, 2' deoxynucleosides represented at A in Figure 4; in Example 12 for the 5'-protected amino nucleosides represented at B in Figure 4; and in Example 13 for the N-protected morpholino-type subunits shown at D in Figure 4. The general reaction conditions used to activate the hydroxyl groups in these structures are generally applicable; subunit activation reactions involving thiocarbonyl-activated subunits are de tailed in Example 14 for the 5'-amino nucleosides represented at B; and in Example 15, for the morpholino-type subunits shown at D in Figure 3.
Following the activation reaction, the activated complex is purified by conventional methods, such as silica gel chromoatography, and then reacted with a second subunit whose selected recognition moiety R2 will form the next in-sequence recognition moiety in the completed polymer. The coupling reaction is preferably carried out under mild conditions in which the activated group can react with backbone N2 amine groups, but not N1 hydroxyl groups. Therefore, the method is suitable for coupling subunits of the type represented by structures B-D in Figure 4, but not subunit structure A. An advantage of this coupling method is that the second subunit -- which contains an amine N1 nucleophile and a hydroxyl N2 nucleophile -- will be coupled to the first activated subunit only through the N1 amine, so it is not necessary to protect the N2 backbone group. The resulting dimer subunits are therefore coupled through an N2-E-N1 bond, as indicated in the figure.
The oligorner can be extended by repeating the above steps of (a) activating the free N2 nucleophile in the second subunit, separating the activated species from the activating agent, and coupling the activated compound with the next-in-sequence subunit, whose backbone is unprotected. This method is used particularly in forming short oligorner blocks by solution methods which will be described below and which are suitable for solid-phase block assembly.
For forming a polymer by solid-phase sequential subunit addition, the second coupling method outlined in Figure 5B is much preferred. The second method differs from the first method in that polymer growth occurs by addition of an excess of activated subunit to an existing subunit or polymer chain, rather than, as in the first method, by addition of an unactivated subunit to an activated chain. In Figure 58, the existing subunit or subunit chain is shown by a subunit whose recognition moiety is R1 (second line in Figure 6B). This subunit has a free N1 backbone nucleophile and an N2 nucleophile which is protected by a preferably acid-stable linkage to a solid support or by virtue of being linked to a chain of subunits which are linked to a solid support. Methods of forming cyclic backbone subunits which are N2 protected in this manner are described generally in Examples 12-15. The first subunit (the most recently added subunit in a growing polymer) is now reacted with an activated subunit which thereby becomes the next-in-sequence subunit in the polymer. This activated subunit, which is activated at its N2 backbone site, and protected at its N1 site, preferably by an acid-labile protective group, is prepared by methods described above with reference to Figure 6.
As seen in Figure 5B, the coupling reaction adds the N2-activated second subunit to the N1-protected first subunit to couple the two through an N2-E-N1 bond, and form a compound which is protected at both free backbone nucleophile sites. This compound is now treated, for example by reaction with acid, to deprotect the acid-labile N1 protective group on last-added subunit, and the procedure is repeated to build up a desired sequence polymer.
It can be appreciated from the above that the
N2-activated subunit which will form the last-in-sequence subunit must be protected at its N1 backbone site, to allow selective activation at the N2 site and to prevent self-polymerization of the activated compound Also the N1-deprotected subunit which is to couple to the activated subunit should be protected at its N2 site to allow selective reaction of its N1 moiety with the N2-activated subunit. Therfore, since the reacting subunits are both protected at one backbone site, the method is suitable for coupling nucleosides (structure A in Figure 4) as well as subunits whose cyclic backbones contain both amine and hydroxyl backbone nucleophiles, such as structures B-D in this figure. The reaction methods used in coupling structure A subunits through a carbonate bond are detailed in Example 12, Briefly, a subunit containing a 5' protected backbone moiety is activated at its 3' hydroxyl, and reacted with another subunit (or growing chain) which is protected at its 3' hydroxyl. The coupling reaction is carried out in the presence of a catalyst, such as N-methylimidazole or N,N-dimethylaminopyridine, which is necessary for forming the carbonate bond. For coupling subunits containing structure B-D cyclic backbones, where intersubunit carbamate or thiocarbarnate bonds are formed, much milder uncatalyzed coupling conditions, such as those described with reference to the first method above, are suitable.
The advantage of this second coupling method, for forming a polymer by solid-phase subunit addition, is that a substantial molar exess of the activated subunit can be added to the growing support-bound polymer at each polymer-addition step, to achieve subunit coupling to a very high percentage of the support-bound polymers. This insures that a large percentage of the support-bound polymers will contain the complete sequence of subunits desired. By contrast in the first method, where the growing polymer is activated at its last-added subunit, the efficiency of subunit addition is limited by the efficiency of the activations steps.
Figure 6 shows the dimeric structures produced by coupling the cyclic backbone subunits indicated at A-A through D-D in Figure 4. The nucleoside subunits in structure A in the figure are joined by a carbonate (Y=O). In the remaining three structures B-D, the subunits are joined by carbamate (Y=O) or thiocarbamate (Y=S) bonds. As seen from the figure, all of the subunit linkages are uncharged and achiral, i.e., do not contain chiral centers. In addition, the linkages are stable in aqueous medium, as judged by the ability of polymers to resist hydrolysis over an extended period in neutral aqueous solution.
According to another important feature of the invention, the structures, when joined to form polymers show acceptible Watson/Crick base pairing with complementary polynucleotides.
Finally, according to another important feature of the invention, polymers formed from the subunits are stereoregular. This is achieved in the structures shown by (a) using natural nucleosides or nucleoside derivatives or synthetic nucleosides having the natural stereoisomeric configuration as subunits, and (b) joining the subunits by achiral intersubunit linkages. Exemplary coupling methods are detailed in Examples 11-15.
B. Subunit Coupling: N1----E Backbone Configurations
General methods for coupling subunits having an
N1-----E backbone configuration are illustrated in
Figures 7A, 7B, and 7C. As will be recalled from Section I above, N1 is a nucleophilic backbone group, such as a hydroxyl or amine group, and E is an electrophile, such as a carboxyl or sulfonyl group, which, when activated, can react with a second
N1-----E type backbone to form an N1-----E-N1 -----E backbone-linked dimer. The subunit backbones of this types which have been described specifically above are the cyclic backbone structμpes E-H in Figure 3, and the acyclic backbone structures I-N in Figure 4. In the cyclic structures E and F, the N1 nucleophile is the
3' hydroxyl group, and in structures G and H, the amine attached to the 3' nitrogen on the morpholino ring. The
E group in all of the cyclic structures is the carboxyl or corresponding sulfonyl group attached to the 5' position of the ribose ring, or to the 6' position of the morpholino ring. In all of the structures shown in
Figure 4, the N1 and E groups are respectively, the backbone amine or hydrazine and carboxylic or sulfonic acid groups on the backbone moiety ends
The first coupling method, which is illustrated in Figure 7A, is analogous to the method described above with reference to Figure 5A, where a first
N1-protected subunit is activated, then coupled directly to a backbone-unprotected subunit which forms the next-in-sequence subunit in the growing polymer. The P1 protective group on the subunit N1 is preferably an acid-labile protective group, such as t-butoxycarbonyl or a clevable linker bound to a solid support. Methods for N.-protecting cyclic backbone structures are analogous to procedures described above for cyclic structures A-D. Similar methods are effective to protect the amine nitrogens in the acyclic bonebone structures. Alternatively, if the polymer is to be constructed on a solid phase support, as described below, the backbone N1 site may be protected by its attachment to a solid support, with subunit addition to the growing polymer occuring via the activated E electrophile of the last-added subunit.
The activation reaction is designed to yield an activated or chain-terminal moiety of the form N1-----E-X, where X is as described above with reference to Figure 6, and the activated subunit has much the same reactivity toward backbone nucleophilic groups as does the activated subunit in the previously described method. That is, the activation is designed to produce much the same active coupling group as in the
N1----N2 type backbones, but where the reactive carbonyl or sulfonyl group is provided by the backbone itself rather than by a carbonyl or sulfonyl activating reagent, as in the methods shown in Figure 6.
The activation can be performed readily by reacting the N1----E type subunit with a carbodimide in the presence of p-nitrophenol, where the backbone electrophile is a carbonyl group, or by reacting with a carbodiimide in the presence of imidazole, 1,2,4 triazole or tetrazole, where the backbone is a sulfonyl. Subunit activation reactions involving carbonyl ele ctrophili c groups are detailed in Examples 16 and 17. The general reaction conditions used to activate the N-amino-morphilino and linear chain backbone carbonyl groups are generally applicable to the other N1----E type backbone structures shown in
Figures 3 and 4.
Following the activation reaction, the activated complex is purified by conventional methods, such as silica chromatography, or, in the case of a support-bound activated chain, simply washed, and then reacted with a second subunit whose selected recognition moiety R2 will form the next in-sequence recognition moiety in the completed polymer. The coupling reaction yields a dimer whose subunits are therefore coupled through an E-N1 bond, as indicated in the figure. As above, both subunits must be suitably base protected during the activation and coupling reactions. The polymer can be extended by repeating the above steps of (a) activating the free E electrophile in the second subunit, (b) separating the activated species from the activating agent, and (c) coupling the activated compound with the next-in-sequence subunit, whose backbone is unprotected.
The second coupling method, which is illustrated in Figure 7B, is directly analogous to the method described above with respect to Figure 5B. Here a first subunit having an E-protected backbone is reacted with a second subunit with an activated E group and a protected N1 nucleophile, to form a dimer linked through an E-N1 bond and protected at both free backbone end groups. Where the polymer is being formed by solid-phase synthesis, the P2 protective "group" takes the form of an acid-stable linkeage to a solid support. The N1-protected subunit is prepared and activated as above. Coupling conditions generally follow those used in the above cyclic subunit coupling reactions.
In the third coupling method, shown at Figure 7C, the n1-protected and E-unprotected subunits (or polymer units) are reacted together in the presence of a suitable E-activating agent, such as a carbodiirnide, as indicated
Figure 8 shows the dimeric structures produced by coupling the cyclic amd acyclic backbone subunits indicated at E-K in Figures 3 and 4, and indicated at E-E through K-K, repsectively. The nucleosides subunits in structure F-F in Figure 3 are joined by an ester linkage. In remaining cyclic backbone structure, G-G, the subunits are joined through a hydrazid or sulfonyl hydrazide linkage. All of the acyclic backbone subunits, shown at I-I, J-J and K-K, are linked through amide (E=carbonyl) or sulfonamide (E=sulfonyl) bonds. As in the above-discussed cyclic backbone subunit structures, all of the subunit linkages are uncharged and achiral, and all of the bonds are reasonably stable in aqueous medium, as judged by the known stability of ester, hydrazide and amide linkages in solution.
According to another important feature of the invention, the structures, when joined to form polymers show Watson/Crick base pairing with complementary polynucleotides.
Finally, as with the N1-----N2 backbone subunits described above, all of the N1----E backbone structures shown are stereoregular. This is due to (a) the homochiral (structures E-H in Figure 3 and I and L in Figure 4) or achiral (structures J, K, M, and N in Figure 4) nature of the recognition moiety attachment to the backbone moiety, and (b) the achiral subunit linkage.
C. Subunit Coupling: Alternating Charged and Uncharged Achiral Linkages
In some applications, it may be desirable to introduce one or more charged achiral subunit linkages in the polymer molecules. The backbone charge may be used to enhance polymer solubility in many solution applications or to adjust polymer-target binding constants, as will be described further below. Polymers having regularly spaced charged achiral backbone linkages, e.g., between every second or third subunit, are suitable for the novel diagnostic system which is described below. For purposes of the present discussion, it is assumed that polymeric units composed of two or more subunits which are internally joined by achiral uncharged linkages are to be linked by a charged achiral linkage. These uncharged units are formed by one of the methods described in Sections A and B above.
The dimer subunits which are to be linked are selected such that that the free N2 group of one dimer can be coupled to the free N1 group of the other dimer through a suitable charged achiral linkage. The preferred linkage is a phosphodiester linkage which requires that the free N2 group on one subunit and the free N1 group on the other subunit both be hydroxyls. As can be seen in Figure 3, this condition is met if dimers formed from either structure A or structure B subunits, which both have a free N2 hydroxyl, are linked to dimers formed from structure A, C, or D subunits which all have a free N1 hydroxyl.
In a typical coupling method, dimer A, which is protected at its N1 position, as indicated, is carried through a series of reactions which lead to an reactive phosphoester coupling species linked to the N2 hydroxyl through a phosphoester bond, as indicated in Figure 9. The preferred coupling method, which is detailed in U.S. patent application for "Polynucleotide Assay Reagent and Method", Serial Number 712,396, filed March 15, 1985, and which follows literature methods for forming phosphodiester bonds between base-protected nucleosides or nucleoside analogue subunits, involves formation of a reactive N1 protected N2-(p-chlorophenyl2-cyanoethyl)-phosphate. The activated dimer is then reacted with the second dimer to form a tetramer which is linked alternately by uncharged and charged achiral intersubunit linkages.
The phosphodiester linkage method may be incorporated, either is solution or support-type polymer synthesis method, according to the general procedures outlined above for polymer coupling involving achiral, but uncharged linkage reactions.
Although for diagnostic purposes, polymers with alternating charged and uncharged achiral bonds may be suitable, it is generally preferred in diagnostic and particularly therapeutic uses, to introduce as few charged linkages as are necessary for achieving adequate polymer solubility in an aqueous medium. The solubility enhancement is generally achieved with 1-3 charged backbone linkages per polymer of about 20 subunits, and preferably no more than about one charged linkage per four subunits. As defined herein, achiral backbone linkages are considered substantially uncharged if they contain less than about one charge backbone linkage per dimeric unit, and preferably one or fewer backbone linkage per tetrameric unit.
IV. Polymer Targeting Considerations
A. Diagnostic Applications
An important application of the uncharged or substantially uncharged polymers of the invention is in a novel solid-support diagnostic system for use in determining the presence and/or amount of a polynucleotide analyte. The assay system includes a solid support reagent composed of a multiple polynucleotide-binding polymers which are constructed according to the invention linked to a solid support.
The polymers are constructed, according to targeting strategies detailed below, for binding specifically to target polynucleotide analyte at a selected melting temperature (Tm). The sytstem also includes polycationic reporter molecules which are designed to bind to the fully charged analyte backbone, but not the uncharged or only partially charged polymer backbone, under selected binding conditions. Each reporter molecules carries one or more reporter groups, and each polynucleotide can accomodate binding of typically several thousand or moe reporter molecules. Thus the system has an ampliication factor, in terms of reporter signal per bound analyte molecule, of several orders or magnitude. The assay system and its method of practice are more fully described in co-owned U.S. Patent application for "Polynucleotide Assay Reagent and Method" , Serial No. 712,396, filed March 15, 1985.
The particular merits of using sequence-specific polynucleotide binding polymers having achiral predominatly uncharges intersubunit linkages for diagnostic applications derives from two factors. First, their target binding is relatively insensitive to salt and so the polymer/target annealing step can be carried out under low salt conditions wherein there is no competitive annealing between the target and complementary nucleic acid sequences in the solution. Second, the polymer's reduced charge or uncharged backbone allows use of the above-described reporter molecules which are designed at high density to the target analyte's polanionic backbone.
The design considerations applied in preparing a polynucleotide binding polymer for use as a diagnostic reagent are governed by the nature of the target analyte and the reaction conditions under which the analyte is to be assayed. As a first consideration, there is selected a non-homopolymeric target base sequence against which the polymer is directed. This target sequence is preferably unique to the analyte being assayed, i.e., is calculated to occur only with some defined small probability (such as 1% or less) in an assay mixture containing a given number of unique-sequence bases. The probability of occurrence of a given n-base target sequence is approximately 1/4n-- that is, a given n-base target sequence would be expected to occur approximately once in a polymer containing 4 bases. Therefore, the probability P that a given n-base sequence will occur in polynucleotides containing a total of N unique-sequence bases is approximately P=N/4n. To illustrate, the probability
P that a 9-base target sequence will be found in a 20 kilobase polynucleotide is about 20x10 3 /2x105 or
0.08, the probability that a 16-base target sequence will be present is about 20x10 3 /4.3x109 or
0.0000047. From these calculations, it can be seen that a polymer having 9-16 recognition moieties specific for a defined 9-16 base target sequence should have high specificity for the target sequence in an assay mixture containing only viral genomes, whose greatest complexities correspond to about 400K of unique-sequence bases.
Similar types of calculations show that a 12 to 16 subunit polymer can provide adequate specificity for a viral or bacterial target sequence in an assay mixture containing viral and bacterial genomic material only (largest genomic sizes about 5,000 kilobases), and that a 16 to 22 subunit polymer can provide adequate specificity for a target sequence in a polynucleotide mixture containing mammalian genomic DNA material (genomic sizes of about 5 billion base pairs of unique-sequence DNA).
The polymer/analyte binding affinity, and particularly the temperature at which the polymer just binds with the target sequence (the melting temperature, or Tm) can be selectively varied according to (a) polymer length, (b) the number of hydrogen bonds that can be formed between the recognition moieties and the corresponding, in-sequence bases of the analyte target sequence, (c) backbone charge density, and (d) a concentration of denaturants, such as formamide, which reduces the temperature of melting. From a number of studies on model homopolymer duplexes, it is known that the melting temperature of oligonucleotide duplexes in the 10 to 20 bp range increases roughly 3°C per additional base pair where the complementary bases are capable of forming two hydrogen bonds, and about 6°C per additional base pair where the complementary bases are capable of forming three hydrogen bonds. Therefore, the length of a target sequence, which is initially selected to insure high binding specificity with the polymer, may be extended to achieve a desired melting temperature with the complementary-base polymer, under selected assay conditions. Also, where the recognition moieties used in constructing the polymer are the standard nucleic acid bases, as illustrated above, the target sequence may be selected to have a high percentage of guanine plus cytosine bases for achieving a relatively high polymer/analyte melting temperature, or a relatively high percentage of adenine plus thymine bases, for achieving a relatively low melting temperature.
The backbone charge density of the polymer must be substantially less than that of the polynucleotide analyte, to allow preferential binding of polycationic reporter molecules to the analyte, under conditions where reporter binding to the polymer does not occur. This requirement is met where the spacing between the adjacent negative charges in the polymer backbone is at least twice as great as the spacing between adjacent charged phosphodiester linkages in the analyte. The charge density of the backbone also has an important effect on the polymer/analyte melting temperature, particularly under low salt conditions, where any charge-charge repulsion between the analyte and polymer backbones can act to lower the temperature at which the two can anneal. In general, therefore, a polymer having a less-charged backbone would be expected to show (1) a higher analyte/polymer melting temperature, and (2) less dependence of the melting temperature on salt concentration. Based on these considerations, an uncharged polymer, such as an all-carbamate-linked or all amide-linked polymer, will allow the analyte/polymer annealing reaction in the diagnostic method to be carried out under a wider range of salt-concentrations than a partially charged polymer, wherein some of the linkages constitute achiral but charged phosphodiester linkages.
B. Therapeutic A ppli cations
Requirement for Adequate Specificity
1. Information Requirements
An ideal therapeutic is one which efficiently inactivates a targeted structure unique to the pathogenic state, but which does not detrimentally affect normal structures in the patient. Thus, sequence-specific gene inactivating agents should only effect inactivation of a targeted genetic sequence which contains an amount of sequence information adequate to assure a high probability of that target sequence being unique to the pathogenic state. For example, an agent which can effect target inactivation by binding to only three or four contiguous bases of its target sequence would invariably also attack many mRNAs required for normal cellular functions. In contrast, an agent which requires binding to 30 or more bases in order to effect target inactivation would have an exceedingly small probability of also attacking non-target messengers involved in normal cellular functions.
A reasonable estimation of the desired amount of sequence information which a gene-inactivating agent should recognize when that agent is targeted against a disease-specific mRNA can be calculated as follows. The human genome contains roughly 5 billion base-pairs of unique-sequence DNA. However, essentially all of this material exists in the duplex state and so is largely unavailable for binding to single-strand-directed polynucleotide-binding agent. Thus, polymer binding is largely restricted to RNA transcripts of the genomic DNA. Of that genomic DNA, something on the order of 1% is transcribed in any given cell type. Further, when all cell types in the patient are considered, it is unlikely that a composite of more than 4% of the entire genome is transcribed in the adult human (i.e., post-embryogenesis). Thus, the sequence complexity of all rna sequences inthe adult human is likely to constitute no more than about 200 million bases -- and probably far fewer.
For a gene-inactivating agent to have an expectation of recognizing no target sequences in a cellular pool of 200 million bases of unique sequence genetic material, it should recognize at least n bases in its target, where n is calculated as 2 x 108 = 4n
- giving a minimal target recognition requirement of 14 bases. This suggests that a gene-inactivating agent recognizing in excessof 14 bases in its target will likely have no targets in cellular pools of normal RNA.. Obviously, as the number ob bases recognized in the target sequence increases over this value, the probability that the agent will recognize no inherent cellular RNA sequences also increases. It is noteworthy that as the number of bases recognized in the target increases linearly, this "safety factor" increases exponentially. For example, below are tabulated the number of bases recognized in a target sequence and the corresponding expected number of targets in a pool of 200 million bases of unique-sequence single-stranded genetic material:
No. of bases recognized Expected no. of targets 12 12.0
14 .75
16 .047
18 .0029
20 .00018
From the above one can conclude that for a sequence-specific gene-inactivating agent destined for therapeutic applications, safety considerations probably mandate that the agent recognize at least 14, and preferably 16 to 20, bases in its target sequence.
2. The Concept of a Minimum Inactivation Length (MIL)
In regard to the amount of sequence information actually "recognized" by a sequence-specific gene-inactivating agent, a sueful concept in this regard is that of a minimum inactivating length (MIL), which we define as the minimum number of contiguous bases of a target sequence to which an agent must bind in order to effect gene inactivation in vivo. Irrespective of whether or not the agnet binds a longer sequence of bases, it is this MIL value which determines the number of bases in the targer "recognized" by that agent -- which, in turn, determines the agent's level of sequence specificity.
To illustrate this concept, suppose a gene-inactivating agent has a 16-subunit length but an MIL value of only 10. Such an agent would be expected to inactivate any genetic sequences containing any of the component 10-contiguous-base target sequences. In a cellular pool comprising 200 million bases of unique-sequence single-stranded genetic material, there are an estimated 1000 such target sequences. Further, because the number of base-pairing moieties in the agent is substantially greater than the MIL value, the agent would also be expected to forma variety of partially mispaired complexes with sequences lacking even the required 10-contiguous-base target sequences -- with some of those rnispaired complexes having sufficient stability to effect inactivation of the participating nontarget genetic sequences.
In contrast, in the case of an agent 16 subunits in length and having an MIL value of 16, by definition, such an agent cannot inactivate nontarget sequences because any partially rnispaired complexes with nontarget sequences would not have sufficient stability to effect gene inactivation -- only perfect 16-base-pair agent/target duplexes would have adequate stability to effect gene inactivation.
In regard to determining actual MIL values, when a series of geπe-inactivation agents, all targeted against the same genetic sequence but varying in number of subunits, are tested in living cells known to contain a functioning single-stranded target sequence, the activity of the targeted gene will show a dramatic reduction at the subunit length corresponding to the MIL value. That is, the MIL value corresponds to that number of subunits just sufficient to block the activity of the targeted genetic sequence.
To illustrate further, below is described an exemplary method for determining the minimum inactivating length for a polynucleotide-binding polymer having any selected backbone linkage type.
First, prepare a regular series of binding polymers having the selected backbone type, targeted against the region 57 to 80 of the rabbit β-globin in mRNA (5'-GUGCAUCUGUCCAGTGAGGAGAAG-3'), and ranging in length from 5 to 24 subunits. Next, following the detailed procedures of Black, Murakimi, and Miller, Biochemistry, 24:6132 (1985), add each of these polymers to a separate portion of a commercial rabbit reticulocyte lysate preparation labeled with [35S]-methionine. Thereafter, add rabbit globin mRNA and incubate 1 hour at 37°C. Work each sample up, fractionate by gel electrophoresis, and guantitate the radioactivity incorporated in β-globin as per Blake et al, above. The minimum inactivating length is that minimum polymer subunit length which effects essentially complete blockage of new β-globin synthesis.
It should be reernphasized that this MIL will vary at small amount depending on the targeted sequence, but in general this relatively simple extracellular method serves to give a quite good approximation of the intracellular MIL for any given backbone type. Once this MIL value has been determined for a selected backbone type, the methods described below for MIL adjustment can be rationally applied for optimizing the MIL for a selected target sequence. 3. Tactics for Adjusting the MIL
As a rule, it is often necessary to vary an agent's target-binding affinity over a substantial range in order to ultimately achieve an acceptable MIL value. To this end, a variety of methods, and combinations thereof, can be used for varying the stability of the agent/target duplex, including:
(i) increasing or decreasing the number of base-pairing moieties in the agent; (ii) selecting a target having a sequence providing greater or lesser stacking contributions (see Tnoco et al (31) for a detailed analysis of the effects of base sequence on duplex stability);
(iii) selecting a target sequence containing a greater or lesser proportion of strong-binding bases (G and C) to weak-binding bases (A and U or T);
(iv) replacing one or more weak-binding base-pairing moieties with stronger-binding moieties having the same Watson/Crick pairing specificities (e.g., a 2,6-diaminopurine moiety can be used instead of adenine to effect a substantially stronger bond to U or T in the target sequence; a 5-bromouracil moiety can be used instead of U or T to effect a substantially stronger bond to a in the target sequence; and a 5-bromocytosine moiety can be used instead of cytosine to effect a somewhat stronger bond to G in the target sequence); or replacing one or more strong-binding base-pairing moieties of the agent with a weaker-binding moiety having the same W/C pairing specificities (e.g., a 2-pyrimidinone moiety can be used instead of cytosine to effect a substantially weaker bond to G in the target sequence; and a 6-thioguanine or hypoxanthine moiety can be used instead of guanine to effect a substantially weaker bond to C in the target sequence); and (v) selecting a backbone structure so as to somewhat inhibit or enhance Watson/Crick pairing to the target sequence.
It should be noted that the first three of the above methods actually comprise target selection procedures, while the latter two methods are effected during synthesis of the agents.
A difficulty with method (i) is that sequence specificity requirement generally preclude reduction of the agent's length to less than about 14 subunits. The difficulty with methods (ii) and (iii) is that other more critical targeting criteria can so severely limit the optimal target region that these two MIL adjustment methods have little latitude in which to achieve a significant effect. Because of these limitations, most MIL adjustments are preferably achieved by methods (iv) and (v).
V. Polymer Assembly Strategies
A. Geometric assembly
After selecting a desired polymer length and recognition moiety sequence, according to factors considered in Section IV, the polymer is assembled, using the general subunit coupling procedures detailed above. One method of polymer assembly involves initial preparation of an appropriate set of dimers, linking selected dimers to form tetramers, linking of these to form octamers, and so on. This method is carried out in solution, substantially according to methods described with reference to Figures 5A and 7A above. It should be noted that all couplings need not necessarily be between oligomers of equal size. For example, often it is desirable in the last coupling to link a hexadecamer with a tetramer to give a 20-mer or to link a hexadecamer with an octomer to give a 24-mer.
A particular merit of this assembly method is that each coupling product is roughly twice the mass of the precursors and so purification of the product of each coupling is a simplified matter. Example 18 below details the the assembly of an 8-subunit carbamate-linked polymer formed by this method. (b)
B. Stepwise Assembly on a Solid Support
One preferred method for polymer synthesis is stepwise assembly on a solid support. Here the first subunit in the polymer is attached through its N2 backbone group to a solid support. Typically, a solid support, such as glass beads derivatized with cleavable, preferably acid-stable, long-chain linkers are employed as the support material, and prepared for attachment of N2 nucleophile, such as a 5' hydroxyl of a nucleoside, as descibed in Example 19. The glass beads are reacted with a subunit which has a preferably acid-labile N 1. protected group, and an activated N2 or E backbone group, as detailed in Section III above. The coupling of various types of subunits to a glass-bead support is described generally in Examples 20-22. After coupling the second subunit (or polymer unit which may be assembled in solution) to the support, any unreacted linker nucleophiles are capped by addition of a suitable capping reagent, such as p-nitrophenyl acetate, and thereafter the support is washed and filtered. The protecting group on the N1 terminal subunit is removed, typically by acid treatment, and after neutralization, the support is then reacted with an excess of the next-in-sequence subunit (or polymer unit) which is activated at its free N2 backbone moiety. The excess of activated subunit maximizes the number of support-bound subunits which are chain-elongated. That is, one feature of the solid support assembly method is the need for high coupling efficiencies at each subunit addition step, and the concentration of added activated subunit is selected to maximize this efficiency. Chain elongation is continued in this manner, with capping of failure sequence, after each subunit addition, until the polymer of the desired length and sequence is achieved. The general method is illustrated in Example 20 for the preparation of a 14-subunit carbonate-linked polymer, in Example 21, for the synthesis of a 19-subunit carbamate-linked polymer, and in Example 22, for the assembly of a 19-subunit amide-linked polymer.
After addition of the last-in-sequence subunit, the polymer may be capped with a suitable charged or uncharged group coupled to the terminal N1 group. If the backbone assembly has been carried out exclusively with uncharged achiral linkages, the capping reagent is preferably a charged group which imparts an end terminal charge to the polymer. After capping the polymer, the polymer is cleaved from the support, e.g., by treatment with a nucleophile, such as ammonium hydroxide, and the polymer is purified, such as by anion exchange chromatography. These post-assembly operations are described in Example 23, for synthesis of a carbonate-linked polymer, in Example 25, for synthesis of a carbamate -linked polymer, and in Example 27, for synthesis of an amide-linked polymer.
For diagnostic applications involving the above solid-support reagent with bound polymer molecules, the assembled, purified polymers are attached to a solid support, such as a cellulose support by known coupling methods, such as those detailed in Example 20. Examples 24, 26, and 28 are illustrative.
The following examples illustrate a variety of subunit synthetic schemes, polymer construction methods, and applications and compositions made and used according to the invention. The examples are in no way intended to limit the scope of the invention.
Example 1
Preparation of ribonucleosides and deoxyribonucleosides
The following nucleosides are obtained from Sigma Chemical Co., St. Louis, MO: deoxyuridine, deoxyguanosine, thymidine, deoxyadenosine, deoxycytidine, 5-bromodeoxyuridine, deoxyinosine, 2,6-diamino-9-(2-deoxy-β-D-erythro-pentofuranosyl)9H-purin e (2,6-diaminopurine deoxyriboside), uridine, guanosine, 5-methyluridine, adenosine, cytidine, 5-bromouridine, inosine.
2,6-Diamino-9-(β-D-ribofuranosyl)-9H-purine (2,6-diaminopurine riboside) is obtained from Pfaltz and Bauer, Inc., Division of Aceto Chemical Co., Inc., Waterbury, CT. The following nucleosides are prepared by the literature methods indicated:
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-2-pyrimidinone (2-hydroxypyrimidine deoxyriboside) is prepared by the method of P. Kohler, E. Uolz, U. Sequin, and C. Tamm in Nucleic Acid Chemistry (L.B. Townsend and R.S. Tipson, eds) John Wiley and Sons, Inc. (1976).
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-methoxy-2-pyrimidinone is prepared by the following procedure: 1-(3',5'-Di-O-benzoyl-2-deoxy-β-D-erythro-pentofu ranosyl)-4-rnethylthio-2-pyrimidinone (prepared as in D. Cech and A. Holy, Collection of Czechoslov Chem Comm (1977) 42:2246) is treated with 200 mL of 0.2M sodium methoxide solution. The resulting solution is allowed to stand overnight at room temperature. This solution is then neutralized with Dowex 50X8 (H+ form) and filtered; the residue is dissolved in water. (100 mL) extracted with ether (2.50 mL) and the aqueous phase evaporated. The residue is an amorphous material which isused directly in succeeding reactions.
2-Amino-9-(2-deoxy-β-D-erythro-pentofuranosyl)-1, 9-dihydro-6H-purine-6-thione (deoxythioguanosine) is prepared by the procedure of R.H. Iwamoto, E.M. Acton, and L. Goodman, J Med Chem (1963) 6:684.
1-(β-D-Ribofuranosyl)-2-pyrimidinone (2-hydroxypyrimidine riboside) is prepared by the procedure of U. Niedballa and H. Uorbruggen, J Org Chem (1974) 39:3668. 1-(2-Deoxy-β-D-ribofuranosyl)-4-methoxy-2-pyrimid inone (2-hydroxypyrimidine deoxyriboside) is prepared by the procedure of R. Wightman and D. Holy, Collection of Czechoslov Chem Comm (1973) 38: 1381.
2-Arnino-9-(β-D-ribofuranosyl)-1,6-dihydro-6H-purine-6-thione (thioguanosine) is prepared by the procedure of J.J. Fox, I. Wempen, A. Hampton and I.L. Doerr, J Amer Chem Soc (1958) 80: 1669.
Example 2 Preparation of Base-Protected Nucleotides
Dimethoxytrityl chloride, N-benzoyladenosine, N-benzoyl-2'-deoxyadenosine, N-benzoylcytidine, N-benzoyl-2'-deoxycytidine and N-isobutyryl-2'-deoxyguanosine are obtained from Sigma Chemicals, St. Louis, Missouri.
9-Fluorenylmethoxycarbonyl chloride (FMOC chloride), trimethylchlorosilane, isobutyric anhydride, 4-nitrobenzoyl chloride and all organic solvents for reactions and chromatography are obtained from Aldrich Chemical Co., Milwaukee, Wisconsin. Silica Gel is obtained from EM Science, Cherry Hill, New Jersey.
2.1 Guanosine The N-2 9-fluorenylmethoxycarbonyl derivative is prepared by the procedure below which is general for the protection of nucleoside amino groups:
Guanosine (1 mmol) is suspended in pyridine (5 mL) and treated with trimethylchlorosilane (5 mmol). After the solution is stirred for 15 minutes
9-fluorenylmethoxycarbonyl chloride (5 mmol) is added and the solution maintained at room temperature for 3 hours. The reaction is cooled in an ice bath and water (1 mL) is added. After stirring for 5 minutes cone. ammonia (1 mL) is added and the reaction stirred for 15 minutes. The solution is evaporated to near dryness and the residue dissolved in chloroform (10 mL). This solution is washed with sodium bicarbonate solution (5 mL, 10%), dried over sodium sulfate and evaporated. The residue is coevaporated several times with toluene and the product chromatographed on silica gel using a gradient of methanol in methylene chloride (0-50%).
N-Isobutyrylguanosine is prepared by the method of Letsinger and Miller, J Amer Chem Soc (1969) 91:3356.
N-2 Acetylguanosine is obtained by the method of C.B. Reese and R.S. Saffhill, J Chem Soc Perkin Trans (1972) 1:2937. 2.2 Deoxyguanosine
The N-2 9-fluorenylmethoxycarbonyl derivative is prepared by the method of J. Heikkla and J. Chattopadhyaya, Acta Chem Scand (1983) B37:263. The N-2 acetyl derivative is obtained from
Research Plus Inc., Bayonne, N.J.
2.3 Deoxyadenosine
The N-6 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Hirnmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583.
N-6 4-Nitrobenzoyl-2'-deoxyadenosine is prepared using the procedure above for FMOC-guanosine except that 4-nitrobenzoyl chloride is substituted for FMOC chloride.
The N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya, Tetrahedron Lett (1981) 22: 3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
2.4 Adenosine The N-6 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Hirnmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583.
N-6 4-Nitrobenzoyladenosine is prepared using the procedure above for FMOC-guanosine except that 4-nitrobenzoyl chloride is substituted for FMOC chloride.
The N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya Tetrahedron Lett (1981) 22:3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
2.5 Deoxycytidine
The N-4 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Himmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583. The N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is, prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya Tetrahedron Lett (1981) 22:3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
2.6 Cytidine
The N-4 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by the method of F. Himmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24: 3583. The N-6 2--(phenylsulfonyl)-ethoxy carbonyl derivative is prepared by the procedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethyl chloroformate (obtained from N. Balgobin, S. Josephson and B. Chattopadhyaya Tetrahedron Lett (1981) 22: 3667) is used as the acylating agent and N-methylimidazole or pyridine is used as the solvent.
2.7 2,6-diaminopurine riboside
The N-2,N-6-bis (9-fluorenylmethoxycarbonyl) derivative of 2,6-diaminopurine riboside is prepared by the general procedure. The N-2 ,N-6-bis (isobutyryl) derivative is prepared by the general procedure.
2.8 2,6-diaminopurine-2'-deoxyriboside The bis N-2,N-6-(9-fluorenylmethoxycarbonyl) derivative of 2,6-diaminopurine-2'-deoxyriboside is prepared by the general procedure.
2.9 Thioguanosine The N-2 9-fluorenylmethoxycarbonyl derivative of thioguanosine is prepared by the general procedure.
2.10 2'-Deoxythioguanosine
The N-2 9-fluorenylmethoxycarbonyl derivative of 2'-deoxythioguanosine is prepared by the general procedure.
Example 3 Preparation of 5'-amino-2',5-dideoxyribonucleoside subunits.
Carbon tetrabromide, sodium azide, P-toluenesulfonyl chloride (tosyl chloride), triphenyl phosphine and 10% palladium on carbon are purchased from Aldrich Chem Co. Lithium azide is obtained from Kodak Laboratory and Specialty Chemicals. 3.1 General Method
The nucleosides employed in this example are thymidine, N6-benzoyl-2'-deoxyadenosine, N4-benzoyl-2'-deoxycytodine, and 2'-deoxyinosine, 2-hydroxy-pyrimidine-2'-deoxyriboside and the N2-N6-bisisobutyryl derivative of 2,6-diamino purine-2'-deoxyriboside (see Example 1). The required dried nucleoside (1 mmol) is weighed into a reaction vessel containing triphenyl phosphine (1.01 mmol) and lithium azide (5 mmol). After the solids are suspended in DMF, carbon tetrabromide (1.0 mmol) is added to the vessel. The solution is stirred at room temperature for 24 h. After quenching the reaction with methanol, the solution is evaporated to dryness. The residue is chromatographed on silica gel eluting with methanol/ chloroform mixtures to afford the desired 5'-azido-2', 5'-deoxynucleoside. The 5'-azido nucleoside (1 mmol) is dissolved in ethanol. Hydrogenation in the presence of 10% palladium on carbon (catalytic amount) under a hydrogen atmosphere (35 psi) occurs over 10 h. The solution is filtered and evaporation under reduced pressure affords a crude solid. The solid is purified by trituration with the appropriate solvent. 3.2 5'-Azidouridine derivativ
The 5'-azido derivative of 5-bromo-2'-deoxy uridine is prepared via the method described above. The 5'-azido-t-bromo-2'-deoxyuridine (1 mmol) is treated with triphenyl phosphine (1.5 mmol) in pyridine at room temperature for 2 h. Concentrated ammonia (1 mL) is added to the reaction vessel. After 14 h the solvent is removed under vacuum. The residue is dissolved in THF and this solution is added to hexanes. The precipitate is collected and the solid triturated with the appropriate solvent to afford the 5'-amino-5-bromo-2'-deoxyuridine.
3.5 5'-Azido guanosine
An alternate preparation of 5'-amino-2', 5'-dideoxyguanosine is to treat the protected 2'-deoxyguanosine (protected at either the N-2-acetyl or the N-2-FMOC derivative) (1 mmol) with tosyl chloride (1.3 mmol) in pyridine at 0°C overnight. The solution is evaporated to dryness and the residue is dissolved in chloroform. The resulting solution is washed twice with aqueous sodium bicarbonate and dried over sodium sulfate. The solvent is evaporated and the residue chromatographed on silica gel eluting with methanol/chloroform mixtures. The resulting tosylate (1 mmol) is treated with sodium azide (6 mmol) in DMF at 80-100°C for a few h. The solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with chlorofotrm/methanol solvent mixtures. The azide is reduced to the desired amine using the above procedure.
Alternate protecting groups for the base nitrogens of 2'-deoxycytidine, 2'-deoxyadenosine, 2'-deoxyguanosine, and 2,6-diaminopurinedeoxyriboside are 2-(phenylsulfonyl) ethoxycarbonyl for 2'-deoxycytidine and 2'-deoxyadenosine and the use of the 9'-fluorenylmethoxycarbonyl (FMOC) to protect 2'-deoxyguanosine and 2,6-diaminopurine deoxyriboside, as in Example 2.
Example 4 Preparation of 3'-amino-2', 3' -dideoxyribonucleoside subunits. Thymidine is transformed to 5'-O-acetyl-3'-azido-2', 3'-dideoxythymidine and this is converted to 3'-azido-2', 3'-dideoxyadenosine and 3'-azido-2', 3'-dideoxyguanosine via the methods of M. Imazawa and F. Eckstein, J Org Chem (1978) 43:3044. The base moiety of the 3'-azidoadenosine is protected as either the benzoyl or the 2-(phenylsulfonyl)ethoxycarbonyl derivative (as shown in Example 1). N-2 of the 3'-azidoguanosine is protected as either the acetyl or FMOC derivative (see Example 1). The reduction of these 3'-azido functions to the 3'-amino-2', 3'-dideoxyribonucleosides is performed as shown in Example 3.
Example 5 Preparation of morpholino-type subunits derived from ribonucleosides.
Ammonium biborate, sodium m-periodate, sodium cyanoborohydride, uridine, N4-benzoyl cytidine, and N6-benzoyl adenosine are obtained from Sigma Chemical Co. N2-isobutyryl guanosine is prepared by the general method of Letsinger and Miller (J Amer Chem Soc. (1969) 91:3356). 2-Phenyl-2-propanol and bis (p-nitrophenyl) carbonate are obtained from Aldrich Chemical Co.
The morpholino derivative of uridine is prepared by dissolving 1 mmol of uridine plus 2 mmol of ammonium biborate, (NH4)2B2O7, in 5 ml of water. While stirring in an ice bath protected from direct light 1.2 mmol of sodium m-periodate in 5 ml of water is added. After 90 min, 0.2 ml of 1,2-propanediol is added and stirring is continued for 10 min at rt. Thereafter, while stirring in a well-ventilated hood, 0.25 g of sodium cyanoborohydride dissolved in 3 ml of water is added and the mixture stirred for 4 hr at rt. Thereafter, the reaction mixture is reduced to an oil under vacuum in a warm water bath, resuspended in a minimal volume of methanol, layered on a silica gel chromatography column, and the column eluted with methanol/1% triethylamine. The morpholino product elutes from the column as a sharp band moving significantly slower than the original ribonucleoside and its oxidation products. The morpholino product has a UV spectrum essentially identical to the parent ribonucleoside, but its mass is lower by 17 daltons (determined by mass spectral analysis using fast atom bombardment activation).
The morpholino nitrogen is next protected by reacting the product with 2-phenylisopropyl phenyl carbonate. The required active carbonate is prepared and reacted with the morpholino subunit essentially by the methods of Sandberg and Ragmarsson (Int J Peptide Protein Res (1974) 6:111). Finally, if desired (depending on the method to be used for subunit assembly into polymers) the hydroxyl at the position corresponding to the original 5' can be activated by dissolving the N-protected subunit in a minimal volume of dry dimethylformamide and adding 2 equivalents of bis (p-nitrophenyl) carbonate plus 0.2 equivalent of triethylamine, N-methylimidazole, or N,N-dimethylamino pyridine. After 3 h at rt, the reactions mixture is reduced in volume under vacuum in a warm water bath. The thick syrup is resuspended in a small volume of dichloromethane and layered on a silica gel column, which is subsequently eluted with a mixture of dichloromethane/ether (1:1 by vol) 0.2% in N,N-dimethylaniline by volume. With this solvent system, the activated product moves substantially slower than p-nitrophenol and bis (p-nitrophenyl) carbonate but much faster than the unreacted starting material. The activated product is readily visualized on silica gel TLC plates simply by exposing to ammonia fumes, whereupon a yellow nitrophenolate ion is generated within one to two minutes. Other ribonucleosides (bases protected where necessary as in Example 2) are converted to their corresponding morpholino derivatives by essentially the same methods -- though varying amounts of methanol must be added to effect dissolution of the protected ribonucleosides before the initial periodate oxidation step.
When the morpholino-type subunits are to be coupled via thiocarbamate linkages, the preferred base-protective groups for the common starting ribonucleosides are: the phenylsulfonylethoxycarbonyl moiety for cytidine and adenosine and the 9-fluorenyl methoxycarbonyl moiety for guanosine.
Example 6
Preparation of 5'-acetic acid-2', 5'-dideoxyribonucleoside subunits.
Triethylamine, pyridine/sulfur trioxide complex, p-nitrophenol, dicyclohexylcarbodiimide, and 40% aqueous dimethylamine are obtained from Aldrich.
3'-O-tert-Butyldimethylsilyl-N-2-(phenylsulfonyl) ethoxycarbonyladenosine (1 mmol), prepared as in Example 12.1, is dissolved in methylene chloride and dimethylsulfoxide (10 mL, 1/1, v/v) at 0°C and triethylamine (3 mmol) and the pyridine/sulfur trioxide complex (3 mmol) is added. The mixture is stirred at this temperature for 1 h, then washed with water, dried over sodium sulfate, and evaporated under reduced pressure. The residue is dissolved in dry pyridine (10 mL) and treated with benzyloxycarbonylmethylenetriphenlyphosphorane (1.5 mmol) for 24 h at 37°C. The mixture is then evaporated to dryness under reduced pressure and the residue dissolved in methylene chloride, washed with dilute HCl and water, then the organic phases are dried over sodium sulfate and solvent removed under reduced pressure. The residue is chromatographed on silica using a gradient of methanol in chloroform (0-20%). The unsaturated ester (1 mmol) from the previous paragraph is dissolved in ethyl acetate/ethanol (40 mL, 1/1, v/v) containing cyclohexadiene (5 mL) and 10% palladium on charcoal (100 mg) and the suspension is vigorously agitated under a nitrogen atmosphere for 5-24 h. The mixture is filtered and the solvents removed under reduced pressure. The residue is chromatographed on silica gel using a gradient of methanol in chloroform (5-50%). To a 0.2-0.5 M solution of the acid from the previous paragraph in ethyl acetate (or methylene chloride) p-nitrophenol is added in about 20% excess. The calculated amount of dicyclohexylcarbodiimide is added to the solution at 0°C. After 0.5 h the mixture is allowed to come to room temperature and was held there for 1 h. The dicyclohexylurea which separated is filtered and washed with solvent. The combined filtrate and washings are evaporated to dryness under reduced pressure. The ester may be used directly for coupling reactions or may be purified by short column chromatography on silica using an isopropanol in chloroform gradient (0-50%).
The analogous series of reactions may be carried out on the other properly protected 2'-deoxynucleosides.
The active ester (1 mmol) is treated with 40% aqueous dimethylamine (2 mmol) in methanol to form the N,N-dimethylamide of the N-protected 3'-O-tert-butyl dimethylsilyl-2', 5'-dideoxyadenosine-5'-acetic acid. The solvents are removed by evaporation under reduced pressure and the residue is desilylated by using the 2M HF/1M tert-butylammonium fluoride (TBAF) reagent described in B.L. Gaffney and R.A. Jones, Tetrahedron Lett (1982) 23: 2257. To the 2M HF/1M tert-butylammonium fluoride in pyridine (1 mmol of TBAF) is added the deprotected nucleoside from the previous paragraph. After stirring for 24 h the reaction is partitioned between methylene and aqueous sodium bicarbonate. The organic layer is washed with water, dried over sodium sulfate, and evaporated to dryness. The residue is purified by chromatography on silica using a gradient of methanol in methylene chloride (5-25%).
Example 7
Preparation of Purine and Pyrimidine Pyrrolidones
Potassium t-butoxide, anisoyl chloride, 6-chloropurine, 10% palladium on charcoal, phthaloyl chloride, 2-amino-6-chloropurine, 20% aqueous tetraethylammonium hydroxide, 25% aqueous trimethylamine, 2-pyrimidinone, N-bromosuccinimide, trifluoroacetic acid, 4-nitrophenol, and N,N-disuccinimydyl carbonate are obtained from Aldrich. Lithium azide is obtained from Eastman Kodak, Rochester, New York. C18 reverse phase silica gel is obtained from Whatman, Hillsboro, Oregon.
7.1 Preparation of (S)-5-[(4-amino-2-oxopyrimidinyl)-methyl]-pyrrolidone (henceforth referred to as the cytosine pyrrolidone)
Cytosine (2.2 mrnol) is dissolved in dimethyl sulfoxide (2 mL) which contained potassium tert-butoxide (2 mmol). This solution is added to a flask which contained (S)-5-(tosyloxymethyl)-2-pyrrolidone (1 mmol, prepared by the procedure of E. Hardegger and H. Ott, Helv Chim Acta (1955) 38:312). After dissolution, the mixture is stirred at 25°C for 6 h. The mixture is neutralized with acetic acid (2 mmol) and evaporated under reduced pressure. The residue is taken up in dimethyl formamide (5 mL) and evaporated under reduced pressure, and this procedure repeated three times.
7.2 Preparation of N-4 anisoylcytosine pyrrolidone The mixture from the previous paragraph is dissolved in pyridine or N-methylimidazole (10 mL) and treated with anisoyl chloride (2.8 mmol) at room temperature. After stirring for 2 h, the mixture is quenched with ice (0.5 g). After 5 min, 1 mL of 29% ammonia was added. After 15 min at rt the solution is dissolved in ethyl acetate (15 mL) and washed twice with brine (15 mL). The washings are combined and washed with ethyl acetate (2 x 30 mL), the combined organic layers dried with sodium sulfate and evaporated under reduced pressure. The residue is coevaporated several times with toluene and the residue chromatographed on silica gel with a gradient of methanol in methylene chloride (0-20%).
7.3 Preparation of 6-chloropurine pyrrolidone
This compound is prepared as for the alkylation of cytosine except that the alkylation is performed on
6-chloropurine and the mixture was heated at 35-95°C for several h. The mixture is cooled to room temperature, neutralized with acetic acid (2 mmol) and evaporated under reduced pressure, shaken with a mixture of 20% methanol/chloroform and 10% sodium bicarbonate. The aqueous layer is washed with chloroform, the combined organic layers aree dried over sodium sulfate and evaporated under reduced pressure. The residue is chromatographed on silica gel using a gradient of methanol in chloroform (0-25%). 7.4 Preparation of 6-azidopurine pyrrolidone
The 6-chloropurine pyrrolidone (1 mmol) is treated with lithium azide (2 mmol) in dimethyl sulfoxide (2 mL) at 30-100°C for several h. At the end of this time the mixture is evaporated under reduced pressure, dissolved in chloroform, washed with 10% sodium bicarbonate, and the organic layer dried over sodium sulfate and concentrated under reduced pressure. The residue is chromatographed on silica using a gradient of methanol in chloroform (0-25%).
7.5 Preparation of adenosine pyrrolidone
The azide from the previous example is hydrogenated by shaking the azide (1 mmol) with 30 pounds of hydrogen pressure in a solution of ethanol (10 mL) containing palladium on charcoal (100 mg, 10% by weight Pd) for 24 h. The solution is filtered through celite and the solvent evaporated. The residue is used directly for the next step.
7.6 Preparation of N-6 phthaloyladenosine pyrrolidone
The phthaloyl group is introduced at the N-6 position by the acylation of the adenosine pyrrolidone (1 mmol) with phthaloyl chloride (1.4 mmol) using the procedure above for the anisoylation of cytidine except that no ammonia was added in the workup.
7.7 Preparation of 2-amino-6-chloroρurine pyrrolidone
The pyrrolidone tosylate is alkylated with 2-amino-6-chloropurine as per the preparation of the 6-chloropurine derivative. 7.8 Preparation of guanine pyrrolidone
The chloropurine pyrrolidone (1 mmol) from the previous paragraph is dissolved in diglyme (10 mL) and 25% aqueous trimethylamine (2 mL). The solution is stirred at rt for 5 h, water (10 mL) is added, and the mixture concentrated to 10 mL under reduced pressure. Acetic acid (2 mL) is added, and the mixture was evaporated under reduced pressure to an oil. The residue which contains tetraethylammonium acetate is used directly for the next step.
7.9 Preparation of N-2 acetylguanine pyrrolidone
Guanine pyrrolidone (1 mmol) from the previous paragraph is reacted with acetic anhydride (5 mL) at reflux for 2 h. The solvent is evaporated and the residue coevaporated with dimethylforrnarnide. The residue is dissolved in methanol (5 mL) saturated with ammonia at 0°C, and the solution is stirred for 2 h at this temperature. The solvent is evaporated under reduced pressure arid the residue purified by chromatography on a short column of silica using a gradient of methanol in chloroform (5-50%).
7.10 Preparation of 2-amino-6-azidopurine pyrrolidone This compound is prepared from the
2-amino-6-chloropurine pyrrolidone by use of the procedure of Example 7.4, except that the reaction requires longer times and was performed at a higher temperature.
7.11 Preparation of 2,6-diaminopurine pyrrolidone
This compound is prepared as for the adenosine pyrrolidone by reduction of the 2-amino-6-azido derivative. 7.12 Preparation of N-2 acetyl N-6 phthaloyl 2,6-diamino purine pyrrolidone
The acetyl group is introduced by the method above for N-2 acetylguanine pyrrolidone except that the methanolic ammonia treatment is carried out for 8 h. The solvents are evaporated and the residue is phthaloylated as for the adenosine derivative.
7.13 Preparation of inosine pyrrolidone 6-Chloropurine pyrrolidone (1 mmol) was converted to the inosine derivative by the method used for the preparation of the guanine pyrrolidone derivative. The compound is purified by chromatography on silica using a gradient of methanol in chloroform (5-25%).
7.14 Preparation of 2-hydroxypyrimidine pyrrolidone
This compound is obtained by the alkylation of 2-pyrirnidinone by the procedure used for the preparation of the 6-chloropurine derivative.
Example 8 Preparation of Purine and Pyrimidine T-BOC Amino Acids
The general procedure described below for the cytosine pyrrolidone may be applied to all properly protected pyrrolidone derivatives. The product, referred to as the t-BOC acid is formed by cleavage of the pyrrolidone ring to an acid and a t-BOC amine.
8.1 Preparation of the cytosine t-BOC acid
N-4-Anisoylcytosine pyrrolidone (1 mmol) is dissolved in methylene chloride (0.5 M solution) containing di-tert-butyl dicarbonate (2 mmol), triethylamine (1 mmol), and dimethylaminopyridine (1 mmol). The solution is stirred for 10 h at rt. The volatiles are removed and the residue purified on silica using a gradient of methanol in chloroform (0-20%). The residue is dissolved in tetrahydrofuran (0.2 M solution) to which is then added lithium hydroxide ( 3 mmol, as a 1 M solution). After 5 h at rt, the solution is neutralized by the addition of 10% acetic acid (3 mmol) and the solvents removed under reduced pressure. The residue was dissolved in 9N ammonia and stirred at room temperature for 1-10 h. The solvent is removed under reduced pressure and the residue purified by chromatography on C18 reverse phase silica gel using water and methanol (or trifluoroethanol) mixtures.
8.2 Preparation of uracil t-BOC acid
This is prepared from the cytosine t-BOC acid by the following procedure:
The cytosine t-BOC acid (1 mmol) is dissolved in aqueous acetic acid and treated with sodium nitrite
(1 mmol) at 4°C. The mixture is stirred for 1 h and evaporated to dryness under reduced pressure. The product is purified by chromatography in C18 reverse phase silica gel using water and methanol (or trifluoroethanol) mixtures.
8.3 Preparation of 5-bromouracil t-BOC acid
This compound is obtained from the uracil t-BOC acid by the following procedure: The uracil t-BOC acid (1 mmol) is dissolved in
DMF (3 mL) and treated with N-bromosuccinimide (1.2 mmol). The solution is allowed to stand at room temperature for 16 h. After removal of the solvents under reduced pressure the product is purified by chromatography on C18 reverse phase silica gel using water and methanol (or trifluoroethanol) mixtures.
8.4 Preparation of N-protected t-BOC acid This general procedure for acylating the exocyclic amino groups of the recognition moiety is also applicable to the protection of the adenosine derivative.
The cytosine t-BOC acid (1 mmol) from the previous paragraph is dissolved in N-methylimidazole or pyridine (5 mL) and treated with chlorotrimethylsilane (5 mmol). After 15 min at room temperature the solution is treated with 2-(4-nitrophenyl)ethylchloroformate (3 mmol, obtained from F. Hirnmelsbach and W. Pfleiderer, Tetrahedron Lett (1983) 24:3583). The reaction iss maintained at room temperature for 8 h, then cooled in an ice bath, and water (1 mL) was added. After 5 min, 1 mL of concentrated ammonia is added and the mixture stirred at rt for 15 min. The reaction is then evaporated to dryness and the residue dissolved in water. The solution is made acidic with 2 M HCl and extracted with chloroform. The combined organic extracts are dried over sodium sulfate and evaporated under reduced pressure. The residue is purified by chromatography on silica using a gradient of methanol in chloroform (5-50%).
For protection of the guanine and 2,6-diaminopurine derivatives, the above procedure was modified so that the reaction was carried out in pyridine and the acylating agent was 9-fluorenylmethyl chloroformate. Example 9 Preparation of Purine and Pyrimidine Amino Acids
The following procedure is applicable to all the derivatives. It removes the t-BOC group and frees the primary amino group of the backbone.
The t-BOC acid (1 mmol) was dissolved in 20% trifluoroacetic acid in methylene chloride (5 mL) and stirred at rt for 30 min. Toluene (3 mL) is added and the solvents are evaporated under reduced pressure to give the amino acid trifluoroacetate salt. This is used directly for coupling reactions.
Example 10
Preparation of Subunits for Assembly of Achiral Acyclic Polynucleotide Analogs
10.1 Preparation of the Cytidine Analog Lithium borohydride, di-tert-butyl dicarbonate and bis (triphenylphosphine) palladium II chloride were obtained from Aldrich Chemical Co., Milwaukee, WI. 2-Amino-5-bromopyriιnidine is prepared as per T. Nishikawa, Chem. Pharm. Bull, 9:38 (1961). 2-amino-5-bromopyrimidine is benzoylated at the exocyclic amino group by the general method in Example 2 using benzoyl chloride. The product benzamide (25 mmole) is added to a 45 mL pressure vessel containing triethylamine (10 mL), benzene (10 mL), and bis(triphenylphosphine) palladium II chloride (0.375 rnmole). The bomb is flushed with argon, sealed, and pressurized to 600 psi with carbon monoxide and then pressurized to 1200 psi with hydrogen. The reaction vessel is heated in an oil bath with stirring at 145°C. after all gas absorption had ceased, the reaction is cooled, and the gases vented slowly. After addition of anhydrous ether, the reaction mixture is filtered and evaporated in vacuo. The product was isolated following chromatography in silica gel using a gradient of isopropanol in methylene chloride (0-20%).
The 2-benzamidopyrimidine-5-carboxaldehyde (1 mmol) from the previous paragraph is dissolved in ethanol (5 mL) and treated with 4-aminobutyric acid (1 mmole) and triethylamine (1 mmol). After stirring for one hour, the, mixture is hydrogenated at 30 psi using 10% palladium on carbon (100 mg). After the absorption of hydrogen has ceased, the reaction is filtered and the solvents removed under reduced pressure. The residue is dissolved in methanol which is saturated with ammonia at 0°C and stirred at room temperature for 10 h. After evapoaration of the solvents, the residue iss purified by chromatography on C18 silica gel using methanol water mixtures buffered to pH 7 with ammonium acetate (0.2 M). The amino acid (1 mmol) from the previous paragraph is dissolved in ethanol (3 mL) containing di-tert-butyl dicarbonate (1 mmol). Triethylamine (1.5 mmol) is added slowly at room temperature. After the evolution of carbon dioxide has ceased, the solvents are removed in vacuo and the residue used directly for the next reaction.
The t-BOC-amino acid from the previous paragraph is acylated on the exocyclic amino group by the general procedure in Example 2 using 2-(phenylsulfonyl)-ethoxycarbonyl chloride. The product may be purified by chromatography on silica gel using a gradient of methanol in chloroform (0-50%).
The 2-(phenylsulfonyl)-ethoxycarbonyl-protected BOC amino acid from the previous paragraph was converted into the p-nitrophenyl or N-hydroxysuccinimidoyl active ester by the procedures in Examples 11.3 and 11.4.
10.2. Preparation of the Uridine Analog Ethyl 6-hydroxynicotinate is prepared as per H.
Gault, J. Gilbert, and D. Briaucourt, C. R . Acad. Sci., Paris, Ser. C, 266:131 (1968). Lithium borohydride is obtained from Aldrich. Manganese dioxide is prepared as per Uereshchagin et al, Zhurnal Arganicheskoi Khimil, 8:1129 (1972). Dowex resin is obtained from Bio-Rad Laboratories, Richmond, CA .
Ethyl 6-hydroxynicotinate (49 mmole) in tetrahydrofuran (10 mL) is added dropwise to a suspension of lithium borohydride (100 mmol) in tetrahydrofuran (THF) (75mLO at room temperature under nitrogen. The viscous mixture is stirred at 25°C for 16 hours. Then, 60 mL of 20% acetic acid is added to the cooled suspension. The THF is evaporated under reduced pressure and the remaining aqueous solution is applied to an ion exchange column (Dowex-50W-X8) to remove lithium ion. The column is washed carefully with 250 mL of water, which is then evaporated. Boric acid is removed from the resulting residue by repeated evaporation with methanol. The product is purified by distillation under reduced pressure.
The benzylic alcohol (66 mmol) prepared in the previous paragraph is dissolved in ether (250 mL) and treated slowly (exothermic) with a slurry of manganese dioxide (54 g) in ether (100 mL). After twelve hours at room temperature, the mixture is filtered, the solvent removed under reduced pressure, and the residue distilled in vacuo to give 6-hydroxynicotinaldehyde. The aldehyde from the previous paragraph is reacted with 4-amino butyric acid as for the preparation of the cytidine analog in Example 11.1
The amino acid from the previous paragraph was converted to the corresponding BOC-derivative as for the preparation of the cytidine analog in Example 10.1
The BOC-amino acid from the previous paragraph iss converted into the p-nitrophenyl or
N-hydroxysuccinimidoyl active ester by the procedures in Example 11.3 and 11.4.
Example 11 Preparation of 5'-protected, 3'-activated 2'-deoxynucleo- sides.
11.1 2'-deoxynucleoside 5'-O-dimethoxytrityl derivatives
The procedure below is general for the preparation of 2'-deoxynucleoside 5'-O-dimethoxytrityl derivatives:
N-2 9-Fluorenylmethoxycarbonyl-2'- deoxyguanosine (1 mmol) is dissolved in anhydrous pyridine (2.0 mL) and kept at 0°C. Dimethoxytrityl chloride (1.2 minol) is added in portions (0.15 mmol) during an 8 hour period. After an additional 2 hours methanol (0.5 mL) was added and the solution is concentrated at reduced pressure. The residue is treated with a mixture of sodium bicarbonate (2 mL, 5%) and methylene chloride (5 mL). The aqueous layer is extracted with methylene chloride (2x5 mL) and the combined organic layers are dried over sodium sulfate and evaporated. The residue is coevaporated several times with toluene and the product is purified by chromatography on silica gel using a gradient of methanol in methylene chloride (0-10%).
11.2 5'-O-dimethoxytrityl-2'-deoxynucleoside 3'-O-(4-nitrophenyl) carbonates.
The preparation of 5'-O-dimethoxytrityl-2-deoxycytidine 3'-O-(4-nitrophenyl) carbonates is described in the following procedure which is general for the preparation of the 4-nitrophenylcarbonates. Bis (4-nitrophenyl) carbonate and N,N-dimethylaminopyridine are obtained from Sigma.
5' O-Dimethoxytrityl-N-2-(4-nitrophenyl)-ethoxycarbonyl-2'-deoxycytidine (1 mmol) is dissolved in anhydrous dimethylformamide (5 mL) at room temperature and treated with bis (4-nitrophenyl) carbonate (2 mmol) and then evaporated under reduced pressure. The residue is dissolved in dimethylformamide (5 mL) and N,N-dimethylaminopyridine (0.1 mmol) is added. The yellow solution is evaporated and allowed to react for 1 h. The reaction mixture is dissolved in chloroform (10 mL) and washed with aqueous HCl (5 mL, 0.1 M). The organic layer is washed twice with aqueous NaOH (5 mL, 0.1 M), water (2 mL), dried over sodium sulfate and evaporated. The residue is applied to a silica gel column and eluted with a gradient of isopropanol in chloroform (0-10%). The fractions containing the product are pooled, evaporated, dissolved in chloroform (10 mL) and the aqueous NaOH washes are repeated to remove traces of 4-nitrophenol. The organic layer is washed with water, dried over sodium sulfate and evaporated. The product is homogeneous by TLC and is used directly for preparation of the dimeric carbonates. For the preparations of the carbonates of nucleosides containing the FMOC group it is sometimes advantageous to use N-methylimidazole as the catalyst rather than N,N-dimethylaminopyridine.
11.3 N-protected t-BOC p-nitrophenyl ester
The following two procedures prepare active esters for coupling of subunits. It is general for all the t-BOC acid derivatives.
To a 0.2-0.5 M solution of the t-BOC acid from Example 8.4 in ethyl acetate (or methylene chloride), p-nitrophenol is added in about 20% excess. The calculated amount of dicyclohexylcarbodiimide is added to the solution at 0°C. After 0.5 h the mixture is allowed to come to rt and was held there for 1 h. The dicyclohexylurea which separated is filtered and washed with solvent. The combined filtrate and washings are evaporated to dryness under reduced pressure. The ester may be used directly for coupling reactions or may be purified by short column chromatography on silica using an isopropanol-in--chloroform gradient (0-50%).
11.4 N-protected t-BOC N-hydroxysuccinimidoyl ester To a solution of the t-BOC acid from Example
8.4 (1 mmol) in acetonitrile (5 mL) is added pyridine (1 ininol) and N,N'-disuccinimidyl carbonate (1 mmol). The solution ia stirred at rt for 3 h. The solvents are removed under reduced pressure and the residue dissolved in chloroform, the organic phase washed with 0.1 M HCl and water, dried over sodium sulfate, and evaporated to dryness. The esters are sufficiently pure to be used directly. Example 12 Formation of a carbonate intersubunit linkage. The preparation of cytidinyl-(3'-5')-cytidine carbonate is described in the procedures below. This is a general procedure for the formation of the carbonate intersubunit linkage and the deprotection of the oligocarbonate. Note that the base protecting groups must be 2-(4-nitrophenyl)ethoxycarbonyl, 2-(phenyl sulfonyl)ethoxycarbonyl, or FMOC. Tert-butyl dimethylsilyl chloride is obtained from Aldrich.
12.1 Preparation of 2'-deoxynucleoside 3' -O-tert-butyldimethylsilyl derivatives.
The preparation of 3' -O-tert-butyldimethylsilyl-N-2-(4-nitrophenyl)ethoxycarbonyl-2'-deoxycytidine is described in the following procedure which is a general procedure for the preparation of 2'-deoxynucleoside-3'-O-tert-butyldimethylsilyl derivatives:
To a stirred solution of 5'-O-dimethoxytrityl-N-4-(4-nitrophenyl)-ethoxycarbonyl-2'-deoxycytidine (1 mmol) and imidazole (3.5 mmol) in dry dimethylformamide or tetrahydrofuran (10 mL) is added dropwise a solution of tert-butyldimethylsilyl chloride (3 mmol) in dry DMF (10 mL). The reaction is stirred for 1 h, quenched with ice, extracted with methylene chloride, washed with water and dried over sodium sulfate. The organic layer is evaporated to dryness under reduced pressure and purified by chromatography over silica gel with methylene chloride as eluant. To a solution of the 5'-O-dimethoxytrityl derivative from the previous paragraph (1 mmol) in methylene chloride (5 mmol) is added a solution of zinc bromide in methylene chloride (10 mL, 1 M) containing methanol (15 % v/v). The reaction mixture is stirred for 30 min, quenched with ice, and extracted with methylene chloride. The organic layer is washed with water, dried over sodium sulfate, and evaporated to dryness under reduced pressure. The product is purified by chromatography over silica gel using a gradient of methanol in methylene chloride (0-10%).
5'-O-dimethoxytrityl-N-2-(4-nitrophenyl)ethoxy carbonyl-2'-deoxycytidine-3'-O-(4-nitrophenyl) carbonate (1.5 mmol) and 3 '-O-tert-butyldimethylsilyl-N-4-(4-nitrophenyl)ethoxycarbonyl-2'-deoxycytidine (1 mmol) are dissolved in dimethylformamide (5 mL) and the solvent removed under reduced pressure. This is repeated two times. The residue is redissolved in dimethylformamide (5 mL) and treated with N,N-dimethylaminopyridine (0.5 mmol) or N-methylimidazole (1 mmol). The solvent is removed by evaporation under reduced pressure and the reaction allowed to sit overnight at rt. The residue is dissolved in chloroform (20 mL) and washed with aqueous HCl (2 mL, 0.1 M), water (2 mL), aqueous NaOH (2 x 2 mL, 0.2 M), water (2 mL), and the organic layer dried over sodium sulfate and evaporated. The residue is purified on silica gel using a gradient of isopropanol in chloroform (0-30%). The isolated product is homogeneous on TLC and by 400 MHz NMR. The residue is desilylated by using the 2 M
HF/1M tert-butylammonium fluoride (TBAF) reagent described in B.L. Gaffney and R.A. Jones, Tetrahedron Lett (1982) 23:2257. To the 2M HF/1M tert-butylammonium fluoride in pyridine (1 mmol of TBAF) is added the deprotected nucleoside from the previous paragraph. After stirring for 24 h, the reaction is partitioned between methylene chloride and aqueous sodium bicarbonate. The organic layer is washed with water, dried over sodium sulfate, and evaporated to dryness. The residue is purified by chromatography on silica using a gradient of methanol in methylene chloride (5-25%).
The 3'-hydroxydinucleoside carbonates are converted to the 3'-(4-nitrophenyl)-carbonates by the method in Example 11. These derivatives may be reacted with the 5'-hydroxyl of a nucleoside or oligonucleoside carbonate to prepare higher order oligonucleoside carbonates.
Example 13 Formation of a carbamate intersubunit linkage. Bis (p-nitrophenyl) carbonate, p-anisoyldiphenyl methyl chloride, N,N-dimethylaniline (DMA), and triethylamine are available from Aldrich Chemical Co. The required 5'-amino-2', 5'-dideoxyribonucleoside (employing acetyl or benzoyl groups for base protection where necessary) (1 mmol) prepared in Example 3 above, is treated with p-anisoyl diphenylmethyl chloride (1.2 mmol) in pyridine. After 12 h the solvent is evaporated and the residue is redissolved in chloroform. This solution is washed once each with aqueous NaHCO3 and water, then dried over Na2SO4. After evaporation of the solvent, the residue is chromatographed on silica gel eluting with chloroform/methanol/ 1% triethylamine mixtures.
The 5'-tritylated aminonucleoside (1 mmol) is evaporated twice from DMF, then treated with bis-(p-nitrophenyl) carbonate (2 mmol) in the presence of triethylamine (or N,N-dimethylaminopyridine, or N-methyl irπidazole) (catalytic amount) using DMF as the solvent. After 3 h, the solvent is evaporated and the residue dissolved in chloroform. This solution is washed twice with 0.01 N aqueous NaOH, once with water, then dried over Na2SO4. The solvent is removed by rotovap and the residue is chromatographed on silica gel eluting first with a chloroform/0.1% DMA mixture, then with a chloroform/methanol/0.1% DMA solvent system. The appropriate fractions are combined and evaporated to dryness. The residue is dissolved in a minimum of THF and this solution is added to an excess of hexanes. The precipitated activated nucleoside is collected by filtration and dried under vacuum.
The required 5'-aminonucleoside (1.1 mmol), as prepared in Example 3 above, is twice evaporated from DMF. The activated nucleoside (1 mmol) is added to the reaction vessel and the solids are dissolved in DMF. The solution is concentrated to a small volume and allowed to stand overnight. The solvent is completely removed under vacuum and the resulting residue is dissolved in chloroform. This solution is twice washed with 0.01 N aqueous sodium hydroxide, once with water, then dried over solid sodium sulfate. The solvent is removed by rotovap, and the residue is chromatographed on silica gel eluting with the appropriate methanol/chloroform/1% triethylamine solvent system. The fractions containing the dimer nucleoside are combined, then evaporated to dryness. The residue is dissolved in a minimum of THF and this solution is added to hexanes. The precipitate is collected and dried under vacuum.
Example 14
Formation of a thiocarbamate intersubunit linkage.
N,N-dimethylaminopyridine (DMAP) is purchased from Aldrich Chemical Co. Formation of the thiocarbamate intersubunit linkage of the 5'-aminonucleosides prepared in Example 3 is achieved in the same manner as described above in Example 13 with the following alterations in procedure. The 5'-aminonucleosides employed have as base-protecting moieties either 2-(phenylsulfonyl)ethoxycarbonyl (for deoxycytidine and deoxyadenosine) or 9-FMOC (for guanosine and 2,6-diamino-2'-deoxyriboside) groups. The preparation of these molecules are described above (Example 3). Activation of the 5'-tritylaminonucleoside (1 mmol) is achieved with p-nitrophenylchlorothioformate (as prepared by G Hilgetag and r. Phillippson, Monatsber Deut Akad Wiss Berlin (1964) 6:897) (1.2 mmol) in the presence of triethylamine (2 mmol) and the DMAP (catalytic amount) in DMF. The coupling step employs this activated monomer (1 mmol) with the requisite 5'-aminonucleoside (1.1 mmol) using DMAP or N-methyl imidazole as catalyst and DMF as the solvent. In this aqueous workup of these steps, the 0.01 N aqueous NaOH washes are omitted, employing in their place water washes.
Example 15 Formation of carbamate and thiocarbamate intersubunit linkages between morpholino type subunits.
N-methylimidazole is purchased from Aldrich Chemical Co.
The requisite dry, N'-protected, 5'-free alcohol morpholino type nucleoside (employing acetyl or benzoyl groups for base protection where necessary) (1 mmol), prepared in Example 5 above is treated with bis-(p-nitrophenyl)carbonate and triethylamine (or DMAP or N-methylimidazole) (catalytic amount) in DMF under anhydrous conditions. The solution is stirred for 3 h, then evaporated to dryness. The residue is dissolved in chloroform and the solution is washed twice with 0.01 N aqueous NaOH, once with water, then dried over Na2SO4. The solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with an appropriate chloroform/isopropanol/0.1% DMA mixture. The fractions containing the desired activated nucleoside are combined, evaporated, and the resulting solid is dissolved in a minimum of THF. The THF solution is added to an excess of hexanes and the resulting precipitate is collected and dried.
The required 5'-free hydroxy, N'-free morpholino type nucleoside (using the identical base protecting groups listed above in this example) (1.1 mmol), after evaporation twice from DMF, is treated with the 5'-(p-nitrophenoxycarbonyl)ιnorpholino subunit (1 mmol) in DMF. The volume of the reaction solution is reduced under vacuum to a small volume. If required, a catalytic amount of N-methylimidazole or triethylamine is added to the reaction vessel. The resulting solution is allowed to stand overnight at rt. The remaining solvent is evaporated, and the residue is dissolved in chloroform. The chloroform solution is washed twice with 0.01 N aqueous NaOH, once with water, and then dried over Na2SO4. The solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with chloroform/methanol solvent mixtures. The fractions containing the desired dimer are combined, then rotovaped to dryness. The residue is dissolved in a minimum of THF and the solution is added to an excess of hexanes. The precipitate is collected and dried under vacuum.
Morpholino subunits linked by thiocarbamate moieties are prepared as related above in this example with the following experimental alterations. The base-protecting groups are either
1-(phenylsulfonyl)ethoxycarbonyl (for deoxycytidine and deoxyadenosine) or 9-FMOC (for deoxyguanosine and 2,6-diamino-2'-deoxyriboside) groups. The preparation of these molecules is described above (Example 5). Activation of the N-protected morpholino nucleoside (1 mmol) is achieved with p-nitrophenylchlorothioformate (1.2 mmol) in the presence of triethylamine (2 mmol) and DMAP or N-methyl imidazole (catalytic amount) in DMF. The coupling step employs this activated monomer (1 mrnol) with the requisite N'-free morpholino nucleoside (1.1 mmol) using DMAP or N-methyl imidazole as catalyst and DMF as the solvent while warming the solution to 35-40°C. In the aqueous workup of these steps, the 0.01 N aqueous NaOH washes are omitted, employing in their place water washes.
Example 16 Preparation of an ester subunit linkage.
N,N-dimethylaminopyridine and N-methylimidazole are obtained from Aldrich.
The coupling of two subunits is performed by reaction of the 5'-N,N-acetamide-3'-hydroxy derivative with the with the 3'-silylated-5'-active ester. These are prepared as in Example 6. The reagents (1 mmol each) are coevaporated several times with dimethylformamide, then dissolved in dimethylformamide (5 mL) and treated with N,N-dimethylaminopyridine (0.2 mmol) or N-methylimidazole (1 mmol). The solvent is removed under reduced pressure and the reaction allowed to stand at room temperature for 2 h. The residue is dissolved in chloroform and washed with dilute HCl, water, and the organic phase dried with sodium sulfate and solvents evaporated under reduced pressure. The residue is purified by chromatography on silica gel using a gradient of methanol in chloroform.
The chain may be elongated by desilylation as in Example 6, and treatment of the resulting 3'-free hydroxy compound with the active ester as in the previous paragraph.
Example 17 Formation of a t-BOC amide-linked dimer
A fully protected t-BOC N-hydroxysuccinimidoyl ester or p-nitrophenyl ester prepared as in Example 11.3 and 11.4 (1 mmol) is dried by several coevaporations with dry dirnethyformamide. The protected amino acid trifluoroacetate salt from Example 9 (1 mmol) is treated similarly. The two components are separately dissolved in dry dimethylformamide (2 mL), mixed, and treated with diisopropylethylamine (1.0 mmol). The solution is stirred at room temperature for 1 h, then the solvent is removed by evaporation under reduced pressure, the residue dissolved in chloroform and washed with dilute HCl. The organic layer is dried over sodium sulfate, evaporated to dryness under reduced pressure, and the residue purified by chromatography on silica gel using a gradient of methanol in chloroform (5-50%).
Alternatively the t-BOC dimer acid may be purified by chromatography on C18 reverse phase silica gel using water and methanol (trifluoroethanol) mixtures.
17.1 Preparation of the t-BOC dimer active ester The acid from the previous paragraph is converted into the N-hydroxysuccinimidoyl ester or p-nitrophenyl ester by application of the general methods in Example 11.3 and 11.4. 17.2 Preparation of the t-BOC dimer amino acid trifluoro acetate
The t-BOC dimer acid is treated with trifluoro acetate as per the general procedure in Example 9.
17.3 Preparation of a t-BOC tetramer acid
The t-BOC dimer active ester and the dimer acid trifluoroacetate are coupled to the t-BOC tetramer acid by the general procedure. Purification is best effected by chromatography on C18 reverse phase silica gel using water and methanol (or trifluoroethanol) mixtures buffered to pH 7.0 with triethylammonium acetate. In this manner chains of any length may be prepared by coupling an active ester with a free amine component.
Example 18 Geometric assembly of an 8-subunit carbamate-linked polymer
DEAE cellulose is purchased from Sigma Chemical Co. Preparative TLC plates are a product of EM Science purchased from UWR Scientific. HPLC equipment, columns, and supplies are available from Beckman Instruments, Inc. The requisite carbamate-linked dimer (0.2 mmol) prepared as described in example 12, is treated with 4 mL of a 1/1/1 mixture of methanol/THF/glacial acetic acid at rt for 12 h. The solvent is removed under vacuum and the residue is taken up in a minimum of THF. The THF solution is added to a large volume of hexanes and the resulting precipitate is collected and dried. The acetate salt is dissolved in a 4/1 THF/ethanol mixture and DEAE cellulose (0.8 mmol of the base) is added to the vessel. After stirring for 20 min, the heterogeneous mixture is filtered and the filtrate evaporated to dryness. The residue is dissolved in a minimum of 4/1 THF/ethanol and this solution is added to hexanes. The solid is collected and dried. The precipitation procedure is repeated once.
The required N-5'-trityl dimer (0.1 mmol) (as prepared in Example 12) is evaporated twice from DMF, then treated with bis (p-nitrophenyl) carbonate (2 mmol) in the presence of triethylamine (or DMAP or N-methyl imidazole) (catalytic amount) using DMF as the solvent. after 3 h the solvent is evaporated and the residue dissolved in chloroform. This solution is washed twice with 0.01 N aqueous NaOH, once with water, then dried over Na2SO4. The solvent is removed by rotovap and the residue is chromatographed on silica gel eluting with a chloroforin/isopropanol or methanol/0.1% DMA mixture. The appropriate fractions are combined and evaporated to dryness. The residue is dissolved in a minimum of THF and this solution is added to an excess of hexanes. The precipitated activated dimer is collected by filtration and dried under vacuum. The 5'-amino dimer nucleoside prepared above
(0.11 mmol) is twice evaporated from DMF. The activated dimer (0.1 mmol) is added to the reaction vessel and the solids are dissolved in DMF. The solution is concentrated to a small volume and allowed to stand overnight. The solvent is completely removed under vacuum and the resulting residue is dissolved in chloroform. This solution is twice washed with 0.01 N aqueous sodium hydroxide, once with water, then dried over sodium sulfate. The solvent is removed by rotovap and the residue is chromatographed on a preparative TLC plate eluting with the appropriate methanol/chloroform/1% triethylamine solvent system. The band containing the tetramer nucleoside is eluted and the solvent is evaporated. The residue is dissolved in a minimum of THF and this solution is added to hexanes. The precipitate is collected and dried under vacuum.
The following transformations are performed using the procedures enumerated above in this example. The requisite N-5'-trityl tetramer nucleoside (0.06 mmol) is treated with 1/1/1 THF/methanol/glacial acetic acid, then purified to afford the 5'-aminotetramer nucleoside. Another aliquot of the N-5'-trityl tetramer nucleoside (0.05 mmol) is activated with bis(p-nitro phenyl) carbonate and subsequently purified. The two tetramers are coupled, worked up, and further purification is achieved either by chromatography on a preparative TLC plate or by HPLC chromatography. the chromatographed material is isolated as a solid, dissolved in a minimum of THF, and then precipitated from hexanes. The octomer nucleoside is collected and dried.
The octamer is configured for further use as follows. The N-5'-trityl octomer nucleoside is treated with 1/1/1 THF/methanol/glacial acetic acid and purified employing the conditions listed above. The 5'-amino octomer nucleoside (0.01 mmol) is treated with succinic anhydride (0.02 mmol) in pyridine at room temperature for two hours. The solvent is removed by rotovap and the residue is evaporated several times from ethanol. The residue is taken up in 3/1 THF/ethanol and added to a 2/1 hexane/benzene mixture. The solid is collected and dried. The N-5'-succinylated octamer nucleoside (0.01 mmol) is treated with a 1/1 mixture of pyridine/concentrated ammonia (1 mL). After standing overnight the solvent is removed by rotovap and the residue is evaporated from ethanol several times. The crude solid is purified using reverse phase (RP-18) HPLC chromatography. The elutants are evaporated and the residue is taken up in DMSO and precipitated from a 1/1 hexane/benzene solvent mixture. The solid is collected and dried.
Example 19 Preparation of a support-bound cleavable linker suitable for stepwise and stepwise/block assembly of polymers: activation of support and attachment of linker.
Long-chain alkylamine-derivatized controlled pore glass (Cat. No. 24875) is obtained from Pierce Chemical Co. 2,2-sulfonyldiethanol and dicyclohexylcarbodiimide are obtained from Aldrich Chemical Co.
The amino groups of the glass support (approximately 40 mmol per gram of support) are reacted with succinic anhydride essentially by the method of Matteucci and Caruthers (J Amer Chem Soc (1981) 103 : 3185) to give free carboxylic acid termini.
One-tenth mol of 2,2'-sulfonyldiethanol (60 % by weight in water) is dried by mixing with three volumes of dimethylformamide (DMF), reducing to a thick syrup under vacuum in a warm water bath, adding a second quantity of DMF, reducing to a thick syrup, and repeating the process a third time. Dry DMF is then added to give a final sulfonyldiethanol concentration of 2 M. A mixture of 1 ml of the sulfonyldiethanol solution plus 0.2 g of dicyclohexylcarbodiimide is added to 1 g of the dry controlled pore glass support carrying carboxylic acid residues and the slurry mixed by rocking or tumbling (not stirring) overnight at rt. Thereafter, the glass support is thoroughly washed with methanol and dried. Example 20 Preparation of a 14-sυbunit carbonate-linked polymer targeted against a conserved seguencein the AIDS viral genome: stepwise assembly on a solid support: addition of the first subunit and chain extension.
N-methylimidazole, 4-(N,N-dirnethylamino) pyridine (DMAP), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are obtained from Aldrich Chemical co. Methyl p-nitrophenyl carbonate is prepared from methyl chloroformate and p-nitrophenol.
The 2'-deoxycytidine subunit(0.1 mmol) prepared as in Example 4.2 (wherein the N4 carries a p-nitrophenethoxycarbonyl moiety, the 5' oxygen carries a di(p-rnethoxy)trityl moiety (DMT), and the 3' oxygen carries a p-nitrophenoxy carbonyl moiety) is dissolved in a minimal volume of dry tetrahydrofuran (THF) or dimethylformamide and added to 0.5 g of thoroughly dried controlled pore glass support carrying a cleavable linker, prepared as in Example 19. Catalyst (either 1 mmol of N-methylimidazole or 0.1 mmol of DMAP) is introduced and the slurry is mixed by rocking or trembling (not stirring) for 2 hours. The slurry is next filtered, and the solid washed thoroughly with THF. Any unreacted hydroxyls are capped by adding two ml of THF 1 M in methyl p-nitrophenyl carbonate and 0.5 M in DMAP and mixing for 20 min at rt. Thereafter, the glass support is washed with THF and filtered. The dirnethoxytrityl at the 5' terminus is then removed by washing the support with dichloromethane followed by treatment with 5 ml of 0.2 M dichloroacetic acid in dichloromethane for 5 min at rt. The glass support is next washed with dichloromethane and filtered. Subsequent subunits, prepared as in Example 1 and activated as in Example 2, are added in a like manner and in the following order: G, A , T, A , A ,C, A ,T,T, T,T,T,C, (G protected with the FMOC moiety, A and C protected with the p-nitrophenethoxy carbonylmoiety).
Example 21 Preparation of a 19-subunit carbamate-linked polymer targeted against a conserved sequence in the AIDS viral genome. Stepwise assembly of oligorner blocks on a solid support: addition of the first subunit and chain extension.
The 5'-amino-2', 5'-dideoxycytidine subunit (0.2 mmol), prepared as in Example 3 (wherein the N4 carries a benzoyl moiety, the 5'-amine carries a p-methoxytrityl moiety, and the 3' oxygen carries a p-nitrophenoxycarbonyl moiety) is dissolved in a minimal volume of dry THF and added to 0.5 g of dried controlled pore glass support carrying a cleavable linker, prepared as in Example 19. Catalyst (either 1 mmol
N-methylimidazole or 0.1 mmol of DMAP) is introduced and the slurry mixed by rocking or tumbling for 2 h. The slurry is next filtered, and the solid washed thoroughly with THF. The mono-p-methoxy trityl is removed by washing with dichloromethane, followed by treatment with 5 ml of 0.2 M dichloroacetic acid in dichloromethane at rt for 1 min. The support is then washed with dichloromethane and filtered. The 5' amino termini are converted to their free amine form by a brief wash with 3 ml of THF containing 1% by volume of diisopropylethylamine, followed by washing with THF and filtration.
0.1 mmol of 3' p-nitrophenoxycarbonyl-activated dimer having the sequence 5'-A-G-3', prepared as in Example 13 (wherein the guanine N2 is protected with an acetyl moiety and the adenine N6 is protected with a benzoyl or a p-nitrobenzoyl moiety), is dissolved in a minimal volume of dry THF and added to the glass support and mixing is carried out for 2 h (no catalyst is added for this and subsequent coupling steps). The slurry is next washed with THF and filtered.
Any unreacted amine moieties are capped by adding 2 ml of THF 2 M in p-nitrophenyl acetate and mixing at rt for 20 min. Thereafter, the glass support is washed with THF and filtered.
The mono-mεthoxytrityl at the 5' terminus is removed with dichloroacetic acid, as before.
Subsequent activated dimeric subunits, prepared as in Example 17 (wherein the exocyclic ring nitrogen of cytosine is protected with a benzoyl moiety and the exocyclic ring nitrogen of A is protected with a benzoyl or a p-nitrobenzoyl moiety), and added in a like manner and in the following order: A-T, C-A , T-A , T-T, T-T, A-C, A-C, C-A (5' to 3').
Subunits having base exocyclic ring nitrogen protective groups removable by strong nonnucleophilic bases (e.g., p-nitrophenethoxycarbonyl or phenylsulfonyl ethoxycarbonyl for A and C, and FMOC for G) can also be used for assembling polymers by the procedure above. However, polymers having these alternative protective groups are generally deprotected by treatment with DBU rather than by ammonium hydroxide. Example 22 Preparation of a 19-subunit amide-linked polymer targeted against a conserved sequence in the AIDS viral genome. Stepwise assembly of oligorner blocks on a solid support: addition of the first subunit and chain extension.
A cytosine-containing acyclic-backboned subunit (0.2 mmol), prepared as in Example 11.3 (wherein the N4 of the cytosine carries a 2(P-nitrophenyl)ethoxycarbonyl moiety, the amine of the backbone carries a t-butoxycarbonyl moiety, and the carboxyl is in the form of a p-nitrophenyl ester) is dissolved in a minimal volume of dry THF and added to 0.5 g of dried controlled pore glass support carrying a cleavable linker, prepared as in Example 19. Catalyst (either 1 mmol of
N-methylimidazole or 0.1 mmol of DMAP) is introduced and the support mixed for 2 h, filtered, and the solid is washed thoroughly with THF.
The t-BOC is removed by washing the support with dichloromethane followed by treatment at rt with 5 ml of 20% by volume trifluoroacetic acid in methylene chloride for 30 min. The support is washed with dichloromethane and filtered. The support-bound amino terminus is converted to the free amine form by a brief wash with 3 ml of THF containing 1% by volume of diisopropylethylamine, followed by a THF wash.
The activated dimeric subunit (0.1 mmol) having the sequence (amino terrninus)-G-A-(carboxy terminus) prepared as in Example 17 (wherein the N2 of guanine carries an FMOC moiety, the N6 of adenine carries a p-nitrophenethoxycarbonyl moiety, the backbone amino terminus carries a t-BOC moiety, and the carboxy terminus is in the form of a p-nitrophenyl ester) is dissolved in a minimal volume of dry THF and added to the glass support and mixing is carried out for 2 h (no catalyst is added for this or subsequent coupling steps). The support is washed with THF.
Any unreacted amine moieties are capped by adding 2 ml of 2 M p-nitrophenyl acetate in THF and mixing at rt for 20 min. Thereafter, the glass support is washed with THF.
The terminal t-BOC moiety is removed with TFA and the support neutralized, as described above. Subsequent dimeric subunits, prepared as in
Example 17, are added in a like manner in the following order: (C-terminus) T-A , A-C, A-T, T-T, T-T, C-A, C-A, A-C (N-terminus).
Subunits having exocyclic ring nitrogen protective groups removable by good nucleophiles (e.g., benzoyl for C, benzoyl or nitrobenzoyl for A, and acetyl or isobutyryl for G) can also be used for assembling polymers by the above procedures. However, polymers having these alternative protective groups are generally deprotected by treatment with ammonium hydroxide rather than by DBU.
Example 23 Configuring the carbonate-linked polymer of Example 20 for a solution application.
The 5' oxygen of the support-bound polymer of Example 20 is succinylated with 5 ml of a THF solution 1 M in succinic anhydride and 0.1 M in DMAP for 10 min at rt, washing with THF, and filtering. Next, the polymer is both released from the support and its recognition moieties are deprotected by treatment with 3 ml of dry DMSO containing 0.3 g of DBU at rt for 12 h. The polymer is freed from DBU by two cycles of precipitation with anhydrous either and resuspension in anhydrous DMSO. Thereafter, the polymer is resuspended in agueous DMSO containing a trace of pH 7.5 buffer to neutralize any residual DBU and purified by anion exchange chromatography.
Example 24
Configuring the carbonate-linked polymer of Example 20 for a solid phase application.
6-Aιninocaproic acid is reacted with 2-phenylisopropylphenyl carbonate by the method of
Sandberg and Ragrnarsson (Int J Peptide Prot Res (1974) 6: 111). The N-protected (10 mmol) product is then activated by reaction with 20 mmol of DCC and 50 mmol of p-nitrophenol in a minimal volume of DMF. the resultant product is purified on silica gel developed with ether. The support-bound polymer of Example 20 is reacted with 1 mmol of the above aminocaproic product plus 0.1 mmol of DMAP in 2 ml of THF for 2 h at rt. The glass support is then washed with THF and filtered the polymer is both released from the support and its recognition moieties deprotected by treatment with 3 ml of dry DMSO containing 0.3 g of DBU at rt for 12 h. The polymer is freed from DBU by two cycles of precipitation with anhydrous ether and resuspension in anhydrous DMSO. The phenylisopropoxycarbonyl group is removed by adding 80% aqueous acetic acid and incubating at rt for 2 h. The deprotected polymer is precipitated with ether, resuspended in aqueous DMSO, and purified by cation exchange chromatography. One gram of cellulose (Type 20, Cat. No. 3504, average particle size of 20 microns, from Sigma Chemical Co.), 1 g of bis (p-nitrophenyl) carbonate, and 0.2 ml of triethylamine are added to 5 ml of DMF and stirred overnight at rt. The slurry is filtered, washed once with 10 ml of DMF, then with 50 ml of methanol, and dried under vacuum. A typical level of activation obtained by this procedure is 23 μmol of p-nitrophenoxycarbonyl moiety bound per gram of cellulose.
The activated cellulose (0.5 g) is washed with DMSO, filtered, and 10 μmol of polymer in 1 ml of DMSO is added followed by 30 μmol of diisopropylethyl arnine. This mixture is stirred slowly overnight at rt in a closed vessel, filtered, and washed with DMSO. The support is next washed with aqueous pyridine, rinsed with methanol, and dried.
Example 25 Configuring the carbamate-linked polymer of Example 21 for a solution phase application.
The 5' amino moiety of the support-bound carbamate-linked polymer of Example 21 is succinylated by reacting with 5 ml of a THF 1 M solution in succinic anhydride and 1 M in diisopropylethylamine for 10 min at rt, then washing with THF.
The polymer is released from the support by treatment with 3 ml of DMF containing 0.3 g of DBU. The polymer is freed from the DBU by 2 cycles of precipitation with ether and resuspension in DMF. Protective groups are removed from the recognition moieties by precipitating the polymers with ether, resuspending in 1 ml of DMSO, and adding 1 ml of concentrated ammonium hydroxide. Generally, when the protective group on adenine is a benzoyl moiety, deprotection is carried out in a closed container at 30°C for 12 h. Alternatively, when the protective group on adenine is a nitrobenzoyl and the protective group on guanine is an acetyl moiety, deprotection is carried out at rt for 6 h. Volatile components are removed under vacuum and the polymer solution neutralized with pH 7.5 buffer. Finally, the polymer, diluted in aqueous DMSO, is purified by anion exchange chromatography.
Example 26
Configuring the carbamate-linked polymer of Example 20 for a solid phase application.
The activated 6-aιninocaproic acid reagent (1 mmol) of Example 23 in 3 ml of THF is reacted with the support-bound carbamate-linked polymer of Example 21 for
20 min at rt. The support is washed with THF and filtered. The terminal pheπylisopropoxy carbonyl moiety is removed by washing with dichloromethane, then reacting with 5 ml of 0.5% trifluoroacetic acid in dichloromethane for 15 min at rt, washing with THF, and filtering.
The polymer is released from the support and protective groups on the recognition moieties are removed as in Example 25, except that the final purification is by chromatography on a cation exchange column.
The purified polymer is linked to a cellulose support as in Example 20.
Example 27
Configuring the amide-linked polymer of Example 22 for a solution phase application.
The support-bound amide-linked polymer of Example 22 is succinylated as in Example 24.
The polymer is released from the support, its protective groups on the recognition moieties are removed, and it is purified as in Example 24. Example 28 Configuring the amide-linked polymer of Example 22 for a solid phase application.
The support-bound amide-linked polymer of Example 22 is reacted with activated aminocaproic acid and the phenylisopropoxycarbonyl moiety removed as in Example 24.
The polymer is released from the support, its protective groups on the recognition moieties are removed, it is purified, and then linked to a cellulose support essentially as in Example 24.
While preferred embodiments of the invention have been described herein, it will be appraent to those in the art that various modifications and changes can be made without departing from the invention.

Claims

IT IS CLAIMED :
1. A polymeric composition effective to bind, with a selected binding affinity, to a single-stranded polynucleotide containing a target sequence of bases, comprising non-homopolymeric, substantially stereoregular polymer molecules of the form:
B ~... B, where:
(a) where R1-Rn are recognition moieties selected from purine, purine-like, pyrimidine, and pyrimidine like heterocycles effective to bind by
Watson/Crick pairing to corresponding, in-sequence bases in the target sequence;
(b) n is such that the total number of Watson/Crick hydrogen bonds formed between a polymer molecule and target seguence is at least about 15;
(c) B B are backbone moieties joined predominantly by chemically stable, substantially uncharged, predominantly achiral linkages;
(d) the backbone moiety length ranges from 5 to 7 atoms if the backbone moieties have a cyclic structure, and ranges from 4 to 6 atoms if the backbone moieties have an acyclic structure; and
(e) the backbone moieties support the recognition moieties at positions which allow Watson/Crick base pairing between the recognition moieties and the corresponding, in-sequence bases of the target sequences.
2. The composition of claim 1, wherein the recognition moieties are selected from the following group:
Figure imgf000101_0001
3. The composition of claim 1, wherein the recognition moieties are selected from the following group:
Figure imgf000101_0002
4. The composition of claim 1, wherein B is a cyclic backbone moiety, and B ~ B has one of the following forms:
Figure imgf000102_0001
5. The composition of claim 4, wherein B ~ B has one of the following forms :
Figure imgf000103_0001
where Y = S, O.
6. The composition of claim 1, wherein B is an ayclic backbone moiety, and B ~ B has one of the following forms:
Figure imgf000103_0002
7. The composition of claim 1, for use in binding to an intracellular target sequence, wherein the polymer molecules contain at most 1-3 ionic charges, and no more that about 1 charge per 4 subunits.
8. The composition of claim 7, wherein the polymer molecules contain charged groups at one or both molecule ends.
9. A method of preparing a substantially uncharged polymeric composition effective to bind specifically and with a substantially uniform binding affinity to a single-stranded polynucleotide, said method comprising (1) selecting a specific sequence of bases in the polynucleotide, said sequence constituting the target sequence;
(2) Providing a group of subunits of the form:
B-Ri, and B-Rj, where:
(a) Ri and Rj are recognition moieties selected from purine, purine-like, pyrimidine, and pyrirnidine-like heterocycles effective to bind by Watson/Crick pairing involving two and three hydrogen bonds, respectively, to corresponding bases contained in the selected target-specific sequence;
(b) B is a backbone moiety which can be coupled to another backbone moiety B by a chemically stable, uncharged, achiral linkage;
(c) the spacing between adjacent backbone moieties, when coupled, is 6 atoms if the backbone moieties have a cyclic structure, and between 4-6 atoms if the backbone moieties have an acyclic structure; and
(d) the backbone moieties, when coupled, form a backbone which supports the recognition moieties at positions which allow Watson/Crick base pairing between the recognition moieties and the corresponding, in-seguence bases of the target sequences; and
(3) coupling the subunits in a defined sequence corresponding to the sequence of bases in the target-specific sequence.
10. The method of claim 9, which further includes providing both Ri and Rj recognition moieties for at least one base in the target-spepcific sequence, and adjusting the binding affinity of the composition for the target sequence by decreasing the ratio of Ri/Rj, to increase the binding affinity, and increasing the ratio, to decrease the binding affinity.
11. The method of claim 9, which further includes introducing a group which carried an ionic charge at or near neutrality at one or both ends of the polymer molecules, to increase the solubility of the polymer in an aqueous medium.
12. The method of claim 9, wherein Ri are selected from the following group :
Figure imgf000105_0001
and Rj are selected from the following group:
Figure imgf000106_0001
13. The method of claim 9, wherein B is a cyclic backbone moiety, and adjacent backbone moieties, when linked together, have one of the following forms:
Figure imgf000106_0002
14. The method of claim 9, wherein B is an acyclic backbone moiety and adjacent backbone moieties, when linked together, have one of the following forms:
Figure imgf000107_0001
15. A method for inhibiting expression of a single-stranded polynucleotide contained in a system in which a selected activity is expressed, said method comprising,
(1)selecting a target sequence in the polynucleotide which is required for such expression,
(2) providing a group of subunits of the form:
B-Ri, where:
(a) Ri are recognition moieties selected from purine, purine-like, pyrimidine, and pyrimidine like heterocycles effective to bind by Watson/Crick pairing involving two or three hydrogen bonds, to corresponding bases contained in the selected target sequence; (b) B is a backbone moiety which can be coupled to another backbone moiety B by a chemically stable, uncharged, achiral linkage;
(c) the unit length of backbone moieties, when coupled, is 5 to 7 atoms if the backbone moieties have a cyclic structure, and 4 to 6 atoms if the backbone moieties have an acyclic structure; and
(d) the backbone moieties, when coupled, form a backbone which supports the recognition moieties at positions which allow Watson/Crick base pairing between the recognition moieties and the. corresponding, in-sequence bases of the target sequences;
(3) coupling the subunits in a defined sequence to form a predominantly uncharged, stereoisomer polymer designed to bind specifically to the target sequence,
(4) additing the polymer to the system and observing the extent of inhibition of the polynucleotide expression, depending on the degree of inhibition of polynucleotide expression which is observed, adjusting the binding affinity of the polymer for the target sequence by one or both of the steps of (a) varying the length of the target sequence and the corresponding polymer length (b) varying the ratio of recognition moeities which form three and two hydrogen bonds with corresponding in-sequence polynucleotide bases; and by said adjusting producing a polymer whose binding affintity is substantially the lowest affinity which is required to effect such inhibition of polynucleotide expression.
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Cited By (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0281390A2 (en) * 1987-03-02 1988-09-07 Gen-Probe Incorporated Polycationic supports for nucleic acid purification, separation and hybridization
FR2627590A1 (en) * 1988-02-24 1989-08-25 Ire Celltarg Sa NUCLEIC ACID PROBE COMPRISING A CHEMICALLY MODIFIED 5 (PREMIUM) (OH) TERMINAL NUCLEOTIDE FOR NON-RADIOACTIVE MARKING AND METHODS OF PREPARATION
US4912199A (en) * 1987-07-06 1990-03-27 The Governors Of The University Of Alberta Oligopeptide anticancer and antiviral agents
EP0411186A1 (en) * 1989-08-01 1991-02-06 Abbott Laboratories DNA probe assay using neutrally charged probe strands
US5126439A (en) * 1987-09-22 1992-06-30 Temple University Artificial dna base pair analogues
EP0506830A1 (en) * 1989-12-20 1992-10-07 Antivirals Inc. Uncharged morpholino-based polymers having phosphorous-containing chiral intersubunit linkages
EP0506845A1 (en) * 1989-12-20 1992-10-07 Antivirals Inc. Uncharged morpholino-based polymers having achiral intersubunit linkages
WO1992020703A1 (en) * 1991-05-24 1992-11-26 Ole Buchardt The use of nucleic acid analogues in diagnostics and analytical procedures
WO1993000352A1 (en) * 1991-06-20 1993-01-07 Antivirals Inc. Sequence-specific binding polymers for duplex nucleic acids
US5185444A (en) * 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
EP0549686A1 (en) * 1990-09-20 1993-07-07 Gilead Sciences, Inc. Modified internucleoside linkages
US5235033A (en) * 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
EP0565646A1 (en) * 1991-04-19 1993-10-20 Baylor College Of Medicine Nucleosides and oligonucleosides with a phosphate-free internucleoside backbone and process for preparing the same
WO1993022326A1 (en) * 1992-04-24 1993-11-11 Beckman Instruments, Inc. Protecting groups useful in oligonucleotide synthesis
WO1994012517A1 (en) * 1992-11-23 1994-06-09 Antivirals Inc. Sequence-specific binding polymers for duplex nucleic acids
EP0618925A1 (en) * 1991-12-24 1994-10-12 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
EP0618923A1 (en) * 1991-12-18 1994-10-12 Glaxo Wellcome Inc. Peptide nucleic acids and their effect on genetic material
WO1994022891A1 (en) * 1993-03-31 1994-10-13 Sterling Winthrop Inc. Oligonucleotides with amide linkages replacing phosphodiester linkages
US5359044A (en) * 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
WO1994025477A2 (en) * 1993-04-26 1994-11-10 Nielsen Peter E Novel peptide nucleic acids
US5378841A (en) * 1989-12-20 1995-01-03 Antivirals Inc. Alpha-morpholino ribonucleoside derivatives and polymers thereof
WO1995001369A1 (en) * 1993-07-02 1995-01-12 Ole Buchardt Double-stranded peptide nucleic acids
US5434257A (en) * 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
WO1995031459A1 (en) * 1994-05-11 1995-11-23 Antivirals Inc. Morpholino-subunit combinatorial library and method
US5470974A (en) * 1985-03-15 1995-11-28 Neu-Gene Development Group Uncharged polynucleotide-binding polymers
WO1996002558A1 (en) * 1994-07-15 1996-02-01 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
WO1996005215A1 (en) * 1994-08-17 1996-02-22 The Perkin Elmer Corporation Solid support reagents for the synthesis of 3'-nitrogen containing polynucleotides
WO1996006189A1 (en) * 1994-08-24 1996-02-29 Isis Pharmaceuticals, Inc. Capillary electrophoretic detection of nucleic acids
WO1996014330A1 (en) * 1994-11-02 1996-05-17 Icnpharmaceuticals Amino acid nucleic acids
EP0746326A1 (en) * 1994-01-11 1996-12-11 Isis Pharmaceuticals, Inc. Oligomeric compounds having nitrogen-containing linkages
US5610289A (en) * 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5614617A (en) * 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5618704A (en) * 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5623049A (en) * 1993-09-13 1997-04-22 Bayer Aktiengesellschaft Nucleic acid-binding oligomers possessing N-branching for therapy and diagnostics
WO1997032888A1 (en) * 1996-03-08 1997-09-12 Isis Pharmaceuticals, Inc. Substituted nucleic acid mimics
US5700922A (en) * 1991-12-24 1997-12-23 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US5705333A (en) * 1994-08-05 1998-01-06 The Regents Of The University Of California Peptide-based nucleic acid mimics(PENAMS)
WO1998003542A1 (en) * 1996-07-24 1998-01-29 Buchardt, Dorte Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5714331A (en) * 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5719262A (en) * 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
EP0839830A1 (en) * 1996-10-30 1998-05-06 Bio-Rad Laboratories, Inc. Polyether nucleic acids
US5766855A (en) * 1991-05-24 1998-06-16 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity and sequence specificity
US5792847A (en) * 1989-10-24 1998-08-11 Gilead Sciences, Inc. 2' Modified Oligonucleotides
US5792608A (en) * 1991-12-12 1998-08-11 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
US5817781A (en) * 1992-06-01 1998-10-06 Gilead Sciences, Inc. Modified internucleoside linkages (II)
US5877160A (en) * 1991-05-31 1999-03-02 Genta Incorporated Compositions and methods of treatment of androgen-associated baldness using antisense oligomers
WO1999011820A1 (en) * 1997-09-02 1999-03-11 Isis Pharmaceuticals, Inc. Compositions and methods for the identification and quantitation of deletion sequence oligonucleotides in synthetic oligonucleotide preparations
WO1999013893A1 (en) * 1997-09-16 1999-03-25 Isis Pharmaceuticals, Inc. Peptide nucleic acids having antibacterial activity
US5955571A (en) * 1994-08-08 1999-09-21 Bayer Aktiengesellschaft Nucleic acid-binding oligomers for therapy and diagnosis
US6063569A (en) * 1995-06-07 2000-05-16 Perseptive Biosystems, Inc. Methods for automated synthesis of PNA-DNA chimeras and compositions thereof
WO2000044897A1 (en) * 1999-01-29 2000-08-03 Avi Biopharma, Inc. Method of treating restenosis by antisense targeting of c-myc
US6121433A (en) * 1990-07-27 2000-09-19 Isis Pharmaceuticals, Inc. Oligomeric compounds having nitrogen-containing linkages
US6133444A (en) * 1993-12-22 2000-10-17 Perseptive Biosystems, Inc. Synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions
US6156501A (en) * 1993-10-26 2000-12-05 Affymetrix, Inc. Arrays of modified nucleic acid probes and methods of use
WO2001016365A1 (en) * 1999-08-30 2001-03-08 Bio-Rad Laboratories, Inc. Poly(ether-thioether), poly(ether-sulfoxide) and poly(ether-sulfone) nucleic acids
US6277603B1 (en) 1991-12-24 2001-08-21 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US6414112B1 (en) 1991-05-24 2002-07-02 Ole Buchardt Peptide nucleic acids having 2,6-diaminopurine nucleobases
US6433134B1 (en) 1998-07-09 2002-08-13 Biocept, Inc. Peptide nucleic acid precursors and methods of preparing same
US6441130B1 (en) 1991-05-24 2002-08-27 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
US6476215B1 (en) * 1997-08-01 2002-11-05 Canon Kabushiki Kaisha Ink jet method of spotting a probe and manufacturing a probe array
EP1258492A1 (en) * 1992-09-24 2002-11-20 Chiron Corporation N-substituted oligomers and methods for their synthesis, e.g. polyglycine bearing nucleic acid bases
US6514693B1 (en) 1995-10-12 2003-02-04 Tetramerics Biotechnology, Inc. Method for detecting multiple copies of a repeat sequence in a nucleic acid molecule
US6559303B1 (en) 1995-01-11 2003-05-06 Isis Pharmaceuticals, Inc. Methods for processing chemical compounds having reactive functional groups
US6670393B2 (en) 1995-06-07 2003-12-30 Promega Biosciences, Inc. Carbamate-based cationic lipids
US6710164B1 (en) 1993-11-22 2004-03-23 Peter E. Nielsen Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US6713602B1 (en) 1991-05-24 2004-03-30 Ole Buchardt Synthetic procedures for peptide nucleic acids
WO2004044137A2 (en) * 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Modified oligomeric compounds and compositions for use in gene modulation
US7034131B2 (en) 2001-01-29 2006-04-25 Bio-Rad Laboratories Inc. Nucleic acid derivatives
US7125945B2 (en) 2003-09-19 2006-10-24 Varian, Inc. Functionalized polymer for oligonucleotide purification
US7129244B2 (en) 2003-09-18 2006-10-31 Conforma Therapeutics Corporation Triazolopyrimidines and related analogs as HSP90-inhibitors
US7223833B1 (en) * 1991-05-24 2007-05-29 Isis Pharmaceuticals, Inc. Peptide nucleic acid conjugates
US7241890B2 (en) 2001-10-30 2007-07-10 Conforma Therapeutics Corporation Purine analogs having HSP90-inhibiting activity
US7262192B2 (en) 2003-04-29 2007-08-28 Pfizer Inc. Substituted pyrazolo[4,3-d]pyrimidines and their use as PDE-5 inhibitors
US7375198B2 (en) 1993-10-26 2008-05-20 Affymetrix, Inc. Modified nucleic acid probes
US7544672B2 (en) 2005-03-30 2009-06-09 Conforma Therapeutics Corporation Alkynyl pyrrolo[2,3-d]pyrimidines and related analogs as HSP90-inhibitors
US7569572B2 (en) 2004-04-07 2009-08-04 Pfizer Inc Pyrazolo[4,3-D]pyrimidines
US7572799B2 (en) 2003-11-24 2009-08-11 Pfizer Inc Pyrazolo[4,3-d]pyrimidines as Phosphodiesterase Inhibitors
US7695902B2 (en) 1996-06-06 2010-04-13 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US7696345B2 (en) 2002-11-05 2010-04-13 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US7812149B2 (en) 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
US8124745B2 (en) 2002-11-05 2012-02-28 Isis Pharmaceuticals, Inc Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US8394947B2 (en) 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
US8569474B2 (en) 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
US9150605B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US9150606B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation

Families Citing this family (314)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5521063A (en) * 1985-03-15 1996-05-28 Antivirals Inc. Polynucleotide reagent containing chiral subunits and methods of use
US5599667A (en) * 1987-03-02 1997-02-04 Gen-Probe Incorporated Polycationic supports and nucleic acid purification separation and hybridization
IL86164A0 (en) * 1987-04-28 1988-11-15 Tamir Biotechnology Ltd Improved dna probes
US6054266A (en) * 1987-12-21 2000-04-25 Applied Biosystems, Inc. Nucleic acid detection with separation
US6011020A (en) * 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6465188B1 (en) * 1990-06-11 2002-10-15 Gilead Sciences, Inc. Nucleic acid ligand complexes
AU3250093A (en) 1991-12-12 1993-07-19 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
US6521601B1 (en) * 1992-04-14 2003-02-18 Signal Pharmaceuticals, Inc. Method and composition for inhibition of viral replication
US6770738B1 (en) * 1992-05-22 2004-08-03 Isis Pharmaceuticals, Inc. Higher order structure and binding of peptide nucleic acids
DE4331011A1 (en) * 1993-09-13 1995-03-16 Bayer Ag C-branched oligomers that bind nucleic acids for therapy and diagnostics
AU3999495A (en) * 1994-10-06 1996-05-02 Buchardt, Dorte Peptide nucleic acid conjugates
US8071737B2 (en) * 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes
CA2223050A1 (en) * 1995-06-07 1996-12-19 Abbott Laboratories Probe masking method of reducing background in an amplification reaction
JP3346976B2 (en) * 1996-03-18 2002-11-18 理化学研究所 How to make a full-length cDNA library
US20040161777A1 (en) * 1996-06-06 2004-08-19 Baker Brenda F. Modified oligonucleotides for use in RNA interference
US20040266706A1 (en) * 2002-11-05 2004-12-30 Muthiah Manoharan Cross-linked oligomeric compounds and their use in gene modulation
US5919638A (en) * 1996-10-08 1999-07-06 Abbott Laboratories Reagents and methods useful for detecting prostate tumors
US20050202499A1 (en) 1996-10-31 2005-09-15 Billing-Medel Patricia A. Reagents and methods useful for detecting diseases of the breast
US20030165971A1 (en) * 1996-10-31 2003-09-04 Billing-Medel Patricia A. Reagents and methods useful for detecting diseases of the breast
US20050153373A1 (en) * 1996-10-31 2005-07-14 Billing-Medel Patricia A. Reagents and methods useful for detecting diseases of the breast
US6060246A (en) * 1996-11-15 2000-05-09 Avi Biopharma, Inc. Reagent and method for isolation and detection of selected nucleic acid sequences
US6172209B1 (en) 1997-02-14 2001-01-09 Isis Pharmaceuticals Inc. Aminooxy-modified oligonucleotides and methods for making same
US6127533A (en) 1997-02-14 2000-10-03 Isis Pharmaceuticals, Inc. 2'-O-aminooxy-modified oligonucleotides
US6576752B1 (en) 1997-02-14 2003-06-10 Isis Pharmaceuticals, Inc. Aminooxy functionalized oligomers
US6046004A (en) * 1997-02-27 2000-04-04 Lorne Park Research, Inc. Solution hybridization of nucleic acids with antisense probes having modified backbones
US6251591B1 (en) 1997-02-27 2001-06-26 Lorne Park Research, Inc. Quantitative method for detecting nucleotide concentration
US20020137904A1 (en) * 1998-03-27 2002-09-26 Patricia A. Billing-Medel Reagents and methods useful for detecting diseases of the gastrointestinal tract
US20050208567A1 (en) * 1997-04-25 2005-09-22 Billing-Medel Patricia A Reagents and methods useful for detecting diseases of the prostate
US5962665A (en) 1997-06-16 1999-10-05 Abbott Laboratories Nucleic acid primers and probes for detecting HIV-1 and HIV-2
US6723560B2 (en) * 1998-10-08 2004-04-20 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
US6472209B1 (en) 1997-10-17 2002-10-29 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
US6989270B1 (en) 1997-10-17 2006-01-24 Mayo Foundation For Medical Education And Research Using polyamide nucleic acid oligomers to engender a biological response
US6326479B1 (en) 1998-01-27 2001-12-04 Boston Probes, Inc. Synthetic polymers and methods, kits or compositions for modulating the solubility of same
US6255050B1 (en) 1998-05-22 2001-07-03 Lorne Park Research, Inc. Dynamic hybridization system
US6673912B1 (en) 1998-08-07 2004-01-06 Isis Pharmaceuticals, Inc. 2′-O-aminoethyloxyethyl-modified oligonucleotides
US6043352A (en) 1998-08-07 2000-03-28 Isis Pharmaceuticals, Inc. 2'-O-Dimethylaminoethyloxyethyl-modified oligonucleotides
US6175004B1 (en) * 1998-09-01 2001-01-16 Isis Pharmaceuticals, Inc. Process for the synthesis of oligonucleotides incorporating 2-aminoadenosine
US7935805B1 (en) 1998-12-31 2011-05-03 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
JP4637368B2 (en) 1999-04-14 2011-02-23 ノバルティス バクシンズ アンド ダイアグノスティックス，インコーポレーテッド Compositions and methods for generating an immune response utilizing alphavirus-based vector systems
AU2041901A (en) 1999-11-09 2001-06-06 Elitra Pharmaceuticals, Inc. Genes essential for microbial proliferation and antisense thereto
US20020022718A1 (en) * 1999-12-23 2002-02-21 Forsyth R. Allyn Genes identified as required for proliferation of E. coli
GB2373500B (en) * 2000-02-04 2004-12-15 Aeomica Inc Methods and apparatus for predicting, confirming, and displaying functional information derived from genomic sequence
US7211381B1 (en) 2000-04-04 2007-05-01 Abbott Laboratories β2 andrenergic polymorphism detection
US6593092B2 (en) 2000-04-04 2003-07-15 Abbott Laboratories Beta 2 adrenergic polymorphism detection
AU5752601A (en) 2000-05-04 2001-11-12 Avi Biopharma Inc Splice-region antisense composition and method
AU2001271873A1 (en) 2000-07-06 2002-01-21 Avi Biopharma, Inc. Transforming growth factor beta (TGF-beta) blocking agent-treated stem cell composition and method
US6867349B2 (en) 2000-07-31 2005-03-15 Regents Of The University Of Minnesota Inhibition of gene expression using polynucleotide analogues
US8071740B2 (en) 2000-11-17 2011-12-06 Vascular Biogenics Ltd. Promoters exhibiting endothelial cell specificity and methods of using same for regulation of angiogenesis
US6838452B2 (en) 2000-11-24 2005-01-04 Vascular Biogenics Ltd. Methods employing and compositions containing defined oxidized phospholipids for prevention and treatment of atherosclerosis
WO2002078638A2 (en) * 2001-03-30 2002-10-10 University Of Massachusetts Morpholino imaging and therapy
WO2002086147A2 (en) * 2001-04-24 2002-10-31 Pharmacia & Upjohn Company Single nucleotide polymorphisms diagnostic for schizophrenia
EP1925672A1 (en) 2001-06-22 2008-05-28 Syngeta Participations AG Abiotic stress responsive polynucleotides and polypeptides
US20030198621A1 (en) 2001-07-05 2003-10-23 Megede Jan Zur Polynucleotides encoding antigenic HIV type B and/or type C polypeptides, polypeptides and uses thereof
CA2634992C (en) 2001-07-05 2012-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof
WO2003020980A2 (en) * 2001-08-28 2003-03-13 Pharmacia & Upjohn Company Single nucleotide polymorphisms diagnostic for schizophrenia
US20040115699A1 (en) * 2001-08-28 2004-06-17 Kaytes Paul S. Single nucleotide polymorphisms diagnostic for schizophrenia
US6852491B2 (en) 2001-09-04 2005-02-08 Abbott Laboratories Amplification and detection reagents for HIV-1
US20030224353A1 (en) * 2001-10-16 2003-12-04 Stein David A. Antisense antiviral agent and method for treating ssRNA viral infection
CA2463816C (en) 2001-10-19 2014-07-08 Vascular Biogenics Ltd. Polynucleotide constructs, pharmaceutical compositions and methods for targeted downregulation of angiogenesis and anticancer therapy
US20040029129A1 (en) * 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms
WO2003042226A2 (en) * 2001-11-09 2003-05-22 Pharmacia & Upjohn Company Single nucleotide polymorphisms in gh-1
US20030170694A1 (en) * 2001-12-21 2003-09-11 Daniel Wall Stabilized nucleic acids in gene and drug discovery and methods of use
US20050037962A1 (en) * 2002-01-18 2005-02-17 Ekker Stephen C. Syndecans and angiogenesis
US20030220249A1 (en) * 2002-02-07 2003-11-27 Hackett Perry B. Factors for angiogenesis, vasculogenesis, cartilage formation, bone formation, and methods of use thereof
US20040038303A1 (en) * 2002-04-08 2004-02-26 Unger Gretchen M. Biologic modulations with nanoparticles
US7026120B2 (en) 2002-04-15 2006-04-11 Abbott Laboratories Probes for detecting tumor cells
US7015317B2 (en) * 2002-05-02 2006-03-21 Abbott Laboratories Polynucleotides for the detection and quantification of hepatitis B virus nucleic acids
US20040249130A1 (en) * 2002-06-18 2004-12-09 Martin Stanton Aptamer-toxin molecules and methods for using same
US20040022727A1 (en) * 2002-06-18 2004-02-05 Martin Stanton Aptamer-toxin molecules and methods for using same
US7807802B2 (en) 2002-11-12 2010-10-05 Abbott Lab Polynucleotides for the amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae
US8853376B2 (en) 2002-11-21 2014-10-07 Archemix Llc Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics
AU2003299441A1 (en) * 2002-12-19 2004-07-14 Centre National De La Recherche Scientifique - Cnrs Nf-hev compositions and methods of use
EP2216407B1 (en) 2003-03-07 2016-01-13 Alnylam Pharmaceuticals, Inc. Therapeutic compositions
WO2004094595A2 (en) 2003-04-17 2004-11-04 Alnylam Pharmaceuticals Inc. MODIFIED iRNA AGENTS
EP1629105B1 (en) 2003-04-29 2010-09-01 AVI BioPharma, Inc. Compositions for enhancing transport and antisense efficacy of nucleic acid analog into cells
US20050026189A1 (en) * 2003-05-29 2005-02-03 Liangsu Wang Microbial operons
WO2005013905A2 (en) * 2003-08-07 2005-02-17 Avi Biopharma, Inc. SENSE ANTIVIRAL COMPOUND AND METHOD FOR TREATING ssRNA VIRAL INFECTION
US20050153935A1 (en) * 2003-09-12 2005-07-14 Iversen Patrick L. Compound and method for treating androgen-independent prostate cancer
US20050222068A1 (en) * 2003-10-23 2005-10-06 Mourich Dan V Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells
EP1712242B1 (en) 2003-12-26 2018-05-30 Masatoshi Hagiwara Method of regulating phosphorylation of sr protein and antiviral agents comprising sr protein activity regulator as the active ingredient
WO2005067646A2 (en) 2004-01-07 2005-07-28 Hitachi Chemical Research Center, Inc. Primers and probes for the detection of hiv
WO2005072527A2 (en) * 2004-01-23 2005-08-11 Avi Biopharma, Inc. Antisense oligomers and methods for inducing immune tolerance and immunosuppression
AU2005252663B2 (en) * 2004-06-03 2011-07-07 Isis Pharmaceuticals, Inc. Double strand compositions comprising differentially modified strands for use in gene modulation
EP2500430B1 (en) 2004-06-28 2017-03-08 The University Of Western Australia Antisense oligonucleotides for inducing exon skipping and methods of use thereof
USRE48960E1 (en) 2004-06-28 2022-03-08 The University Of Western Australia Antisense oligonucleotides for inducing exon skipping and methods of use thereof
US8314072B2 (en) 2004-07-02 2012-11-20 Avi Biopharma, Inc. Antisense antibacterial method and compound
US7729761B2 (en) 2004-07-14 2010-06-01 Cardiac Pacemakers, Inc. Method and apparatus for controlled gene or protein delivery
EP1791567B1 (en) 2004-08-10 2015-07-29 Alnylam Pharmaceuticals Inc. Chemically modified oligonucleotides
JPWO2006025154A1 (en) * 2004-08-30 2008-05-08 国立大学法人岐阜大学 Modified oligonucleotide
US8129352B2 (en) * 2004-09-16 2012-03-06 Avi Biopharma, Inc. Antisense antiviral compound and method for treating ssRNA viral infection
US8357664B2 (en) 2004-10-26 2013-01-22 Avi Biopharma, Inc. Antisense antiviral compound and method for treating influenza viral infection
CA2585672A1 (en) 2004-11-01 2006-05-11 Novartis Vaccines And Diagnostics Inc. Combination approaches for generating immune responses
US7524829B2 (en) * 2004-11-01 2009-04-28 Avi Biopharma, Inc. Antisense antiviral compounds and methods for treating a filovirus infection
US20090118132A1 (en) * 2004-11-04 2009-05-07 Roche Molecular Systems, Inc. Classification of Acute Myeloid Leukemia
CA2596506C (en) 2005-02-09 2021-04-06 Avi Biopharma, Inc. Antisense composition and method for treating muscle atrophy
US20060240032A1 (en) * 2005-03-31 2006-10-26 Hinrichs David J Immunomodulating compositions and methods for use in the treatment of human autoimmune diseases
US20080269076A1 (en) * 2005-05-06 2008-10-30 Applera Corporation Multiple Capillary Device and Method for Synthesis and Dispensing
US8067571B2 (en) 2005-07-13 2011-11-29 Avi Biopharma, Inc. Antibacterial antisense oligonucleotide and method
WO2007009094A2 (en) 2005-07-13 2007-01-18 Avi Biopharma, Inc. Antisense antibacterial method and compound
US7790694B2 (en) * 2005-07-13 2010-09-07 Avi Biopharma Inc. Antisense antibacterial method and compound
US7868161B2 (en) * 2005-07-29 2011-01-11 North Carolina State University Photocrosslinking probes and uses of the same
US20100015604A1 (en) 2005-08-17 2010-01-21 Evriklia Lianidou Composition and method for determination of ck19 expression
WO2007030691A2 (en) * 2005-09-08 2007-03-15 Avi Biopharma, Inc. Antisense antiviral compound and method for treating picornavirus infection
US8329668B2 (en) * 2005-09-08 2012-12-11 Avi Biopharma, Inc. Antisense antiviral compound and method for treating picornavirus infection
US8906609B1 (en) 2005-09-26 2014-12-09 Arrowhead Center, Inc. Label-free biomolecule sensor based on surface charge modulated ionic conductance
WO2007047749A1 (en) 2005-10-18 2007-04-26 Novartis Vaccines And Diagnostics Inc. Mucosal and systemic immunizations with alphavirus replicon particles
US8501704B2 (en) 2005-11-08 2013-08-06 Sarepta Therapeutics, Inc. Immunosuppression compound and treatment method
DE602007006457D1 (en) * 2006-03-07 2010-06-24 Avi Biopharma Inc ANTIVIRAL ANTISENSE COMPOUND AND METHOD FOR THE TREATMENT OF ARENA VIRUS INFECTION
RU2394915C2 (en) * 2006-03-24 2010-07-20 Александр Борисович Четверин Non-contact methods of detecting molecular colonies, sets of reagents and device for realising said methods
HUE036995T2 (en) * 2006-05-10 2018-08-28 Sarepta Therapeutics Inc Oligonucleotide analogs having cationic intersubunit linkages
US8785407B2 (en) * 2006-05-10 2014-07-22 Sarepta Therapeutics, Inc. Antisense antiviral agent and method for treating ssRNA viral infection
US20070265215A1 (en) * 2006-05-11 2007-11-15 Iversen Patrick L Antisense restenosis composition and method
US7592009B2 (en) * 2006-10-10 2009-09-22 Ecole Polytechnique Federale De Lausanne (Epfl) Polypeptide ligands for targeting cartilage and methods of use thereof
ES2798758T3 (en) 2007-06-06 2020-12-14 Sarepta Therapeutics Inc Soluble her2 and her3 splice variant proteins, splice exchange oligonucleotides and their use in the treatment of disease
CA2691673A1 (en) 2007-06-29 2009-01-08 Avi Biopharma, Inc. Tissue specific peptide conjugates and methods
AU2008340355B2 (en) 2007-12-04 2015-01-22 Tekmira Pharmaceuticals Corporation Targeting lipids
AU2008345033B2 (en) 2007-12-28 2014-04-03 Sarepta Therapeutics, Inc. Immunomodulatory agents and methods of use
JP5788312B2 (en) 2008-04-11 2015-09-30 アルニラム ファーマスーティカルズ インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． Site-specific delivery of nucleic acids by combining targeting ligands with endosomal degradable components
US8962580B2 (en) 2008-09-23 2015-02-24 Alnylam Pharmaceuticals, Inc. Chemical modifications of monomers and oligonucleotides with cycloaddition
ES2714787T3 (en) 2008-10-24 2019-05-30 Sarepta Therapeutics Inc Exon skipping compositions for DMD
AU2009313201B2 (en) 2008-11-10 2016-06-16 Arbutus Biopharma Corporation Novel lipids and compositions for the delivery of therapeutics
CA2746508A1 (en) 2008-12-17 2010-07-15 Avi Biopharma, Inc. Antisense compositions and methods for modulating contact hypersensitivity or contact dermatitis
EP2403863B1 (en) 2009-03-02 2013-08-28 Alnylam Pharmaceuticals Inc. Nucleic acid chemical modifications
JP5548852B2 (en) * 2009-05-29 2014-07-16 国立大学法人東京農工大学 Hydrophobic group-bonded nucleoside, hydrophobic group-bonded nucleoside solution, and hydrophobic group-bonded oligonucleotide synthesis method
US9200276B2 (en) 2009-06-01 2015-12-01 Halo-Bio Rnai Therapeutics, Inc. Polynucleotides for multivalent RNA interference, compositions and methods of use thereof
PL3431076T3 (en) 2009-06-10 2022-01-31 Arbutus Biopharma Corporation Improved lipid formulation
AU2010279572B2 (en) * 2009-08-03 2017-02-02 Recombinetics, Inc. Methods and compositions for targeted gene modification
CA2780741C (en) 2009-10-12 2023-04-04 Smith Holdings, Llc Methods and compositions for modulating gene expression using oligonucleotide based drugs administered in vivo or in vitro
BR122023023194A2 (en) 2009-11-12 2023-12-26 The University Of Western Australia ANTISENSE OLIGONUCLEOTIDE, COMPOSITION AND RESPECTIVE USE
JP5991922B2 (en) 2009-11-13 2016-09-14 サレプタ セラピューティクス， インコーポレイテッド Antisense antiviral compounds and methods for treating influenza virus infection
EP3296398A1 (en) 2009-12-07 2018-03-21 Arbutus Biopharma Corporation Compositions for nucleic acid delivery
CA2784568A1 (en) 2009-12-18 2011-06-23 Martin A. Maier Lipid particles for delivery of nucleic acids
WO2011100131A2 (en) 2010-01-28 2011-08-18 Alnylam Pharmacuticals, Inc. Monomers and oligonucleotides comprising cycloaddition adduct(s)
WO2011094580A2 (en) 2010-01-28 2011-08-04 Alnylam Pharmaceuticals, Inc. Chelated copper for use in the preparation of conjugated oligonucleotides
WO2011100460A2 (en) * 2010-02-11 2011-08-18 Ecole Polytechnique Federale De Lausanne Ccr7 ligand delivery and co-delivery in immunotherapy
WO2011112516A1 (en) 2010-03-08 2011-09-15 Ico Therapeutics Inc. Treating and preventing hepatitis c virus infection using c-raf kinase antisense oligonucleotides
US9068185B2 (en) 2010-03-12 2015-06-30 Sarepta Therapeutics, Inc. Antisense modulation of nuclear hormone receptors
US9102938B2 (en) 2010-04-01 2015-08-11 Alnylam Pharmaceuticals, Inc. 2′ and 5′ modified monomers and oligonucleotides
EP2558069A1 (en) 2010-04-13 2013-02-20 Novartis AG Benzonapthyridine compositions and uses thereof
WO2011133868A2 (en) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. Conformationally restricted dinucleotide monomers and oligonucleotides
WO2011133876A2 (en) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising acyclic and abasic nucleosides and analogs
WO2011133871A2 (en) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. 5'-end derivatives
WO2011139714A2 (en) 2010-04-26 2011-11-10 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of cysteinyl-trna synthetase
US8961960B2 (en) 2010-04-27 2015-02-24 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of isoleucyl tRNA synthetases
EP2563911B1 (en) 2010-04-28 2021-07-21 aTyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of alanyl trna synthetases
AU2011248490B2 (en) 2010-04-29 2016-11-10 Pangu Biopharma Limited Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Asparaginyl tRNA synthetases
US9034320B2 (en) 2010-04-29 2015-05-19 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Valyl-tRNA synthetases
US8981045B2 (en) 2010-05-03 2015-03-17 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetases
EP2566495B1 (en) 2010-05-03 2017-03-01 aTyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-trna synthetases
JP5976638B2 (en) 2010-05-03 2016-08-23 エータイアー ファーマ， インコーポレイテッド Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of arginyl tRNA synthetase
US9062302B2 (en) 2010-05-04 2015-06-23 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex
US9050373B2 (en) 2010-05-13 2015-06-09 The Charlotte-Mecklenburg Hospital Authority Pharmaceutical compositions comprising antisense oligonucleotides and methods of using same
CA3090304A1 (en) 2010-05-13 2011-11-17 Sarepta Therapeutics, Inc. Antisense modulation of interleukins 17 and 23 signaling
EP2568996B1 (en) 2010-05-14 2017-10-04 aTyr Pharma, Inc. Therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-beta-trna synthetases
US9034598B2 (en) 2010-05-17 2015-05-19 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases
AU2011258106B2 (en) 2010-05-27 2017-02-23 Pangu Biopharma Limited Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutaminyl-tRNA synthetases
CA2799501C (en) 2010-05-28 2022-02-15 Sarepta Therapeutics, Inc. Oligonucleotide analogues having modified intersubunit linkages and/or terminal groups
WO2011149564A1 (en) 2010-05-28 2011-12-01 Tetris Online, Inc. Interactive hybrid asynchronous computer game infrastructure
US9192661B2 (en) 2010-07-06 2015-11-24 Novartis Ag Delivery of self-replicating RNA using biodegradable polymer particles
EP2593125B1 (en) 2010-07-12 2017-11-01 aTyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-trna synthetases
US20130202652A1 (en) 2010-07-30 2013-08-08 Alnylam Pharmaceuticals, Inc. Methods and compositions for delivery of active agents
US20130323269A1 (en) 2010-07-30 2013-12-05 Muthiah Manoharan Methods and compositions for delivery of active agents
US8198429B2 (en) 2010-08-09 2012-06-12 Avi Biopharma, Inc. Antisense antiviral compounds and methods for treating a filovirus infection
US9029506B2 (en) 2010-08-25 2015-05-12 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-tRNA synthetases
US10017763B2 (en) 2010-09-03 2018-07-10 Sarepta Therapeutics, Inc. dsRNA molecules comprising oligonucleotide analogs having modified intersubunit linkages and/or terminal groups
AU2011326364B2 (en) 2010-11-12 2016-08-11 Sarepta Therapeutics, Inc. Antisense antibacterial compounds and methods
AU2012207606B2 (en) 2011-01-11 2017-02-23 Alnylam Pharmaceuticals, Inc. Pegylated lipids and their use for drug delivery
CN103501793A (en) 2011-02-08 2014-01-08 夏洛特-梅克伦堡医院(商业用名:卡罗来纳保健系统) Antisense oligonucleotides
US9528124B2 (en) 2013-08-27 2016-12-27 Recombinetics, Inc. Efficient non-meiotic allele introgression
US10920242B2 (en) 2011-02-25 2021-02-16 Recombinetics, Inc. Non-meiotic allele introgression
WO2012150960A1 (en) 2011-05-05 2012-11-08 Avi Biopharma, Inc. Peptide oligonucleotide conjugates
US20140227293A1 (en) 2011-06-30 2014-08-14 Trustees Of Boston University Method for controlling tumor growth, angiogenesis and metastasis using immunoglobulin containing and proline rich receptor-1 (igpr-1)
CA2840989A1 (en) 2011-07-06 2013-01-10 Novartis Ag Immunogenic combination compositions and uses thereof
WO2013006842A2 (en) 2011-07-06 2013-01-10 Novartis Ag Immunogenic compositions and uses thereof
AU2012284259A1 (en) 2011-07-15 2014-03-06 Sarepta Therapeutics, Inc. Methods and compositions for manipulating translation of protein isoforms from alternative initiation start sites
US9801948B2 (en) 2011-09-21 2017-10-31 Yale University Antimicrobial compositions and methods of use thereof
EP2760477B1 (en) 2011-09-27 2018-08-08 Alnylam Pharmaceuticals, Inc. Di-aliphatic substituted pegylated lipids
US20130085139A1 (en) 2011-10-04 2013-04-04 Royal Holloway And Bedford New College Oligomers
EP2780299A4 (en) 2011-11-18 2015-04-22 Sarepta Therapeutics Inc Functionally-modified oligonucleotides and subunits thereof
ES2832531T3 (en) 2011-11-30 2021-06-10 Sarepta Therapeutics Inc Oligonucleotides for the treatment of repeat expansion diseases
JP6496549B2 (en) 2011-11-30 2019-04-03 サレプタ セラピューティクス， インコーポレイテッド Induced exon inclusion in spinal muscular atrophy
US10465042B2 (en) 2011-12-02 2019-11-05 Yale University Poly(amine-co-ester) nanoparticles and methods of use thereof
WO2013082529A1 (en) 2011-12-02 2013-06-06 Yale University Enzymatic synthesis of poly(amine-co-esters) and methods of use thereof for gene delivery
US9895451B2 (en) 2011-12-02 2018-02-20 Yale University Formulations for targeted release of agents to low pH tissue environments or cellular compartments and methods of use thereof
SI2788487T1 (en) 2011-12-08 2018-09-28 Sarepta Therapeutics, Inc. Oligonucleotide analogues targeting human lmna
EA031110B1 (en) 2012-03-20 2018-11-30 Сарепта Терапьютикс, Инк. Boronic acid conjugates of oligonucleotide analogues
JP6375289B2 (en) 2012-04-05 2018-08-15 マサチューセッツ インスティテュート オブ テクノロジー Immune stimulating composition and method of use thereof
EP2869842A1 (en) 2012-07-06 2015-05-13 Novartis AG Immunogenic compositions and uses thereof
US10111962B2 (en) 2012-09-25 2018-10-30 Genzyme Corporation Peptide-linked morpholino antisense oligonucleotides for treatment of myotonic dystrophy
AU2013364158A1 (en) 2012-12-20 2015-07-09 Sarepta Therapeutics, Inc. Improved exon skipping compositions for treating muscular dystrophy
WO2014113540A1 (en) 2013-01-16 2014-07-24 Iowa State University Research Foundation, Inc. A deep intronic target for splicing correction on spinal muscular atrophy gene
CN105163755A (en) 2013-03-12 2015-12-16 犹他大学研究基金会 Compositions and methods for inducing apoptosis
US20140329881A1 (en) 2013-03-14 2014-11-06 Sarepta Therapeutics, Inc. Exon skipping compositions for treating muscular dystrophy
JP2016521119A (en) 2013-03-14 2016-07-21 サレプタ セラピューティクス， インコーポレイテッド Exon skipping composition for treating muscular dystrophy
EP2968991A2 (en) 2013-03-15 2016-01-20 Sarepta Therapeutics, Inc. Improved compositions for treating muscular dystrophy
EP3708184A1 (en) 2013-03-27 2020-09-16 The General Hospital Corporation Methods and agents for treating alzheimer s disease
US11236338B2 (en) 2013-09-05 2022-02-01 Sarepta Therapeutics, Inc. Antisense-induced exon2 inclusion in acid alpha-glucosidase
SG10201913950XA (en) 2013-09-11 2020-03-30 Eagle Biologics Inc Liquid protein formulations containing viscosity-lowering agents
DK3044315T3 (en) 2013-09-11 2019-05-20 Synthena Ag NUCLEAR INSURANCE AND PROCEDURES FOR TREATMENT OF PUMPS DISEASE
US9708360B2 (en) 2013-09-30 2017-07-18 Geron Corporation Phosphorodiamidate backbone linkage for oligonucleotides
CN105814214A (en) 2013-10-25 2016-07-27 家畜改良有限公司 Genetic markers and uses therefor
WO2015175977A2 (en) 2014-05-16 2015-11-19 Geller Bruce L Antisense antibacterial compounds and methods
US10391098B2 (en) 2014-05-19 2019-08-27 Board Of Regents, The University Of Texas System Antisense antibacterial compounds and methods
TWI787678B (en) 2014-05-23 2022-12-21 美商健臻公司 Inhibiting or downregulating glycogen synthase by creating premature stop codons using antisense oligonucleotides
GB201410693D0 (en) 2014-06-16 2014-07-30 Univ Southampton Splicing modulation
ES2752761T3 (en) 2014-07-24 2020-04-06 Abbott Molecular Inc Compositions for the detection and analysis of mycobacterium tuberculosis
SG10201902499VA (en) 2014-09-03 2019-04-29 Genesegues Inc Therapeutic nanoparticles and related compositions, methods and systems
EP3194581A4 (en) 2014-09-15 2018-04-25 Children's Medical Center Corporation Methods and compositions to increase somatic cell nuclear transfer (scnt) efficiency by removing histone h3-lysine trimethylation
AU2015325055B2 (en) 2014-10-01 2021-02-25 Eagle Biologics, Inc. Polysaccharide and nucleic acid formulations containing viscosity-lowering agents
US20170304459A1 (en) 2014-10-10 2017-10-26 Alnylam Pharmaceuticals, Inc. Methods and compositions for inhalation delivery of conjugated oligonucleotide
WO2016081621A1 (en) 2014-11-18 2016-05-26 Yale University Formulations for targeted release of agents under low ph conditions and methods of use thereof
US10682422B2 (en) 2014-11-18 2020-06-16 Yale University Formulations for targeted release of agents under low pH conditions and methods of use thereof
AU2015372560B2 (en) 2014-12-31 2021-12-02 Board Of Regents, The University Of Texas System Antisense antibacterial compounds and methods
CN107106694B (en) * 2015-01-13 2020-09-22 西格亚有限公司 Process for the preparation of butyric esters of hyaluronic acid sodium salt in water
MA41795A (en) 2015-03-18 2018-01-23 Sarepta Therapeutics Inc EXCLUSION OF AN EXON INDUCED BY ANTISENSE COMPOUNDS IN MYOSTATIN
US20180126014A1 (en) 2015-04-15 2018-05-10 Yale University Compositions for enhancing delivery of agents across the blood brain barrier and methods of use thereof
CA2986228A1 (en) 2015-05-19 2016-11-24 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
EP3302497A4 (en) 2015-06-01 2019-01-16 Sarepta Therapeutics, Inc. Antisense-induced exon exclusion in type vii collagen
WO2016196897A1 (en) 2015-06-04 2016-12-08 Sarepta Therapeutics, Inc. Methods and compounds for treatment of lymphocyte-related diseases and conditions
WO2017011565A1 (en) 2015-07-14 2017-01-19 Abbott Molecular Inc. Compositions and methods for identifying drug resistant tuberculosis
WO2017035278A1 (en) 2015-08-24 2017-03-02 Halo-Bio Rnai Therapeutics, Inc. Polynucleotide nanoparticles for the modulation of gene expression and uses thereof
CN108738310A (en) 2015-09-30 2018-11-02 萨勒普塔医疗公司 Method for treating muscular dystrophy
MA45819A (en) 2015-10-09 2018-08-15 Sarepta Therapeutics Inc COMPOSITIONS AND METHODS FOR TREATING DUCHENNE MUSCLE DYSTROPHY AND RELATED DISORDERS
US10370667B2 (en) 2015-11-18 2019-08-06 Rosalind Franklin University Of Medicine And Science Antisense compounds targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of parkinsons disease
EP3377629B1 (en) 2015-11-18 2023-07-05 Rosalind Franklin University of Medicine and Science Antisense compounds targeting leucine-rich repeat kinase 2 (lrrk2) for the treatment of parkinsons disease
WO2017106304A1 (en) 2015-12-15 2017-06-22 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
AU2016379399B2 (en) 2015-12-23 2022-12-08 Board Of Regents, The University Of Texas System Antisense antibacterial compounds and methods
EP3394262A4 (en) 2015-12-23 2019-12-25 Oregon State University Antisense antibacterial compounds and methods
WO2017143042A2 (en) 2016-02-16 2017-08-24 Yale University Compositions for enhancing targeted gene editing and methods of use thereof
JP2019512014A (en) 2016-02-26 2019-05-09 イェール ユニバーシティーＹａｌｅ Ｕｎｉｖｅｒｓｉｔｙ Compositions and methods of using piRNA in cancer diagnostics and therapeutics
US20210189062A1 (en) 2016-03-01 2021-06-24 Alexion Pharmaceuticals, Inc. Biodegradable activated polymers for therapeutic delivery
MA45328A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF
MA45469A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc BETA-CATENIN NUCLEIC ACIDS AND THEIR USES
WO2017173453A1 (en) 2016-04-01 2017-10-05 The Brigham And Women's Hospital, Inc. Stimuli-responsive nanoparticles for biomedical applications
MA45471A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc PHOSPHATIDYLINOSITOL-3-KINASE NUCLEIC ACIDS AND THEIR USES
MA45470A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc KRAS NUCLEIC ACIDS AND THEIR USES
MA45349A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc EGFR NUCLEIC ACIDS AND THEIR USES
EP3445405A4 (en) 2016-04-18 2019-12-18 Sarepta Therapeutics, Inc. Antisense oligomers and methods of using the same for treating diseases associated with the acid alpha-glucosidase gene
AU2017258642B2 (en) 2016-04-29 2023-08-31 Sarepta Therapeutics, Inc. Oligonucleotide analogues targeting human LMNA
WO2017197128A1 (en) 2016-05-11 2017-11-16 Yale University Poly(amine-co-ester) nanoparticles and methods of use thereof
MA45362A (en) 2016-05-24 2019-04-10 Sarepta Therapeutics Inc PROCESSES FOR THE PREPARATION OF MORPHOLINO OLIGOMERS OF PHOSPHORODIAMIDATE
WO2017205496A1 (en) 2016-05-24 2017-11-30 Sarepta Therapeutics, Inc. Processes for preparing oligomers
CA3024456A1 (en) 2016-05-24 2017-11-30 Sarepta Therapeutics, Inc. Processes for preparing phosphorodiamidate morpholino oligomers
US11472824B2 (en) 2016-05-24 2022-10-18 Sarepta Therapeutics, Inc. Processes for preparing phosphorodiamidate morpholino oligomers
SG11201809499UA (en) 2016-05-24 2018-12-28 Sarepta Therapeutics Inc Processes for preparing phosphorodiamidate morpholino oligomers
MX2018014129A (en) 2016-05-24 2019-04-29 Sarepta Therapeutics Inc Pharmaceutical composition comprising eteplirsen.
JP7069135B2 (en) 2016-06-15 2022-05-17 ユニヴァーシティー オブ ユタ リサーチ ファウンデーション Compositions and Methods for Using Albumin-Based Nanopharmaceuticals
CN109152792B (en) 2016-06-30 2021-09-14 萨勒普塔医疗公司 Process for preparing phosphoric acid diamide morpholino oligomer
KR20190024977A (en) 2016-06-30 2019-03-08 사렙타 쎄러퓨틱스 인코퍼레이티드 Exon skipping oligomer for myopathies
US10683292B2 (en) 2016-07-25 2020-06-16 Nerviano Medical Sciences S.R.L. Purine and 3-deazapurine analogues as choline kinase inhibitors
EP3515445A4 (en) 2016-09-20 2020-05-27 The Regents of The University of Colorado, A Body Corporate Synthesis of backbone modified morpholino oligonucleotides and chimeras using phosphoramidite chemistry
WO2018112470A1 (en) 2016-12-16 2018-06-21 The Brigham And Women's Hospital, Inc. Co-delivery of nucleic acids for simultaneous suppression and expression of target genes
CN110337308B (en) 2016-12-19 2023-09-01 萨勒普塔医疗公司 Exon-skipping oligomer conjugates for muscular dystrophy
IL267246B2 (en) 2016-12-19 2023-03-01 Sarepta Therapeutics Inc Exon skipping oligomer conjugates for muscular dystrophy
SG10202012839TA (en) 2016-12-19 2021-01-28 Sarepta Therapeutics Inc Exon skipping oligomer conjugates for muscular dystrophy
SG11201906200WA (en) 2017-01-06 2019-08-27 Avidity Biosciences Llc Nucleic acid-polypeptide compositions and methods of inducing exon skipping
CN110536694A (en) 2017-04-20 2019-12-03 Atyr 医药公司 For treating pulmonary inflammatory composition and method
GB201711809D0 (en) 2017-07-21 2017-09-06 Governors Of The Univ Of Alberta Antisense oligonucleotide
NL2019390B1 (en) 2017-08-04 2019-02-21 Univ Leiden Screening Method
EP3665202A1 (en) 2017-08-09 2020-06-17 Massachusetts Institute Of Technology Albumin binding peptide conjugates and methods thereof
JP7065951B2 (en) 2017-09-22 2022-05-12 ジュビラント エピパッド エルエルシー Heterocyclic compound as a PAD inhibitor
EA201991450A1 (en) 2017-09-22 2019-12-30 Сарепта Терапьютикс, Инк. OLIGOMER CONJUGATES FOR EXONISM SKIP IN MUSCULAR DYSTROPHY
US20200254002A1 (en) 2017-09-28 2020-08-13 Sarepta Therapeutics, Inc. Combination therapies for treating muscular dystrophy
EP3687547A1 (en) 2017-09-28 2020-08-05 Sarepta Therapeutics, Inc. Combination therapies for treating muscular dystrophy
JP2020536058A (en) 2017-09-28 2020-12-10 サレプタ セラピューティクス， インコーポレイテッド Combination therapy to treat muscular dystrophy
WO2019071028A1 (en) 2017-10-04 2019-04-11 Avidity Biosciences Llc Nucleic acid-polypeptide compositions and uses thereof
US11555189B2 (en) 2017-10-18 2023-01-17 Sarepta Therapeutics, Inc. Antisense oligomer compounds
AU2018352142B2 (en) 2017-10-18 2022-08-25 Jubilant Epipad LLC Imidazo-pyridine compounds as PAD inhibitors
US11629135B2 (en) 2017-11-06 2023-04-18 Jubilant Prodell Llc Pyrimidine derivatives as inhibitors of PD1/PD-L1 activation
GB201718804D0 (en) * 2017-11-14 2017-12-27 Nuclera Nucleics Ltd Novel use
US10953036B2 (en) 2017-11-20 2021-03-23 University Of Georgia Research Foundation, Inc. Compositions and methods of modulating HIF-2A to improve muscle generation and repair
SG11202004537UA (en) 2017-11-24 2020-06-29 Jubilant Episcribe Llc Heterocyclic compounds as prmt5 inhibitors
AU2018378812A1 (en) 2017-12-06 2020-07-09 Avidity Biosciences, Inc. Compositions and methods of treating muscle atrophy and myotonic dystrophy
EP3724335A1 (en) 2017-12-11 2020-10-21 Rosalind Franklin University of Medicine and Science Antisense compounds targeting leucine-rich repeat kinase 2 (lrrk2) for the treatment of parkinsons disease
WO2019139997A1 (en) 2018-01-09 2019-07-18 Trucode Gene Repair, Inc. Polymer-based nanoparticles, related formulation methods, and apparatus
KR20200131845A (en) 2018-03-13 2020-11-24 주빌런트 프로델 엘엘씨 Bicyclic compounds as inhibitors of PD1/PD-L1 interaction/activation
US10765760B2 (en) 2018-05-29 2020-09-08 Sarepta Therapeutics, Inc. Exon skipping oligomer conjugates for muscular dystrophy
EP3806868A4 (en) 2018-06-13 2022-06-22 Sarepta Therapeutics, Inc. Exon skipping oligomers for muscular dystrophy
US10857174B2 (en) 2018-07-27 2020-12-08 United States Government As Represented By The Department Of Veterans Affairs Morpholino oligonucleotides useful in cancer treatment
TW202020153A (en) 2018-07-27 2020-06-01 美商薩羅塔治療公司 Exon skipping oligomers for muscular dystrophy
US20210315820A1 (en) 2018-07-30 2021-10-14 Trucode Gene Repair, Inc. Lipid nanoparticle formulations comprising nucleic acid mimics
EP3830278A4 (en) 2018-08-01 2022-05-25 University of Georgia Research Foundation, Inc. Compositions and methods for improving embryo development
US20210317461A1 (en) 2018-08-09 2021-10-14 Verseau Therapeutics, Inc. Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof
WO2020051507A1 (en) 2018-09-06 2020-03-12 The Broad Institute, Inc. Nucleic acid assemblies for use in targeted delivery
EA202191601A1 (en) 2018-12-13 2022-01-19 Сарепта Терапьютикс, Инк. OLIGOMER CONJUGATES FOR EXON SKIP IN MUSCULAR DYSTROPHY
IL297818A (en) 2018-12-21 2023-01-01 Avidity Biosciences Inc Anti-transferrin receptor antibodies and uses thereof
WO2020157760A1 (en) 2019-01-31 2020-08-06 Bar Ilan University Neoantigens created by aberrant-induced splicing and uses thereof in enhancing immunotherapy
MA55515A (en) 2019-03-28 2022-02-09 Sarepta Therapeutics Inc METHODS OF TREATING MUSCULAR DYSTROPHY WITH CASIMERSEN
WO2020214763A1 (en) 2019-04-18 2020-10-22 Sarepta Therapeutics, Inc. Compositions for treating muscular dystrophy
US11814464B2 (en) 2019-04-29 2023-11-14 Yale University Poly(amine-co-ester) polymers and polyplexes with modified end groups and methods of use thereof
EP3980436A4 (en) 2019-06-06 2023-12-20 Avidity Biosciences, Inc. Nucleic acid-polypeptide compositions and uses thereof
US20220296633A1 (en) 2019-06-19 2022-09-22 Sarepta Therapeutics, Inc. Methods for treating muscular dystrophy
WO2021025899A1 (en) 2019-08-02 2021-02-11 Sarepta Therapeutics, Inc. Phosphorodiamidate morpholino oligomer pharmaceutical compositions
JP2022546699A (en) 2019-08-30 2022-11-07 イェール ユニバーシティー Compositions and methods for delivering nucleic acids to cells
US20230057461A1 (en) 2020-01-27 2023-02-23 The U.S.A., As Represented By The Secretary, Department Of Health And Human Services Rab13 and net1 antisense oligonucleotides to treat metastatic cancer
WO2021163281A1 (en) 2020-02-12 2021-08-19 Accutar Biotechnology Inc. Antisense oligonucleotides and their use for treating pendred syndrome
CA3172111A1 (en) 2020-03-19 2021-09-23 Barbora MALECOVA Compositions and methods of treating facioscapulohumeral muscular dystrophy
EP4126066A1 (en) 2020-03-27 2023-02-08 Avidity Biosciences, Inc. Compositions and methods of treating muscle dystrophy
JP2023531444A (en) 2020-06-19 2023-07-24 イェール ユニバーシティー Poly(amine-co-ester) polymers with modified end groups and enhanced pulmonary delivery
EP4204002A1 (en) 2020-08-31 2023-07-05 Yale University Compositions and methods for delivery of nucleic acids to cells
WO2022140535A1 (en) 2020-12-23 2022-06-30 Sarepta Therapeutics, Inc. Compositions comprising exon skipping oligonucleotide conjugates for treating muscular dystrophy
IL307937A (en) 2021-04-30 2023-12-01 Sarepta Therapeutics Inc Treatment methods for muscular dystrophy
GB202107553D0 (en) 2021-05-27 2021-07-14 Univ Oxford Innovation Ltd Method
GB202111250D0 (en) 2021-08-04 2021-09-15 Univ Oxford Innovation Ltd Method
WO2023034515A2 (en) 2021-09-03 2023-03-09 Sarepta Therapeutics, Inc. Delivery of anitsense oligomers by mirror image peptides
AU2022345098A1 (en) 2021-09-16 2024-04-04 Avidity Biosciences, Inc. Compositions and methods of treating facioscapulohumeral muscular dystrophy
CA3233242A1 (en) 2021-09-30 2023-04-06 Sarepta Therapeutics, Inc. Antisense oligonucleotides having one or more abasic units
WO2023070086A1 (en) 2021-10-22 2023-04-27 Sarepta Therapeutics, Inc. Morpholino oligomers for treatment of peripheral myelin protein 22 related diseases
WO2023159189A1 (en) 2022-02-18 2023-08-24 Yale University Branched poly(amine-co-ester) polymers for more efficient nucleic expression
WO2023168427A1 (en) 2022-03-03 2023-09-07 Yale University Compositions and methods for delivering therapeutic polynucleotides for exon skipping
WO2023178230A1 (en) 2022-03-17 2023-09-21 Sarepta Therapeutics, Inc. Phosphorodiamidate morpholino oligomer conjugates
WO2024015924A2 (en) 2022-07-15 2024-01-18 Entrada Therapeutics, Inc. Hybrid oligonucleotides
WO2024064237A2 (en) 2022-09-21 2024-03-28 Sarepta Therapeutics, Inc. Dmd antisense oligonucleotide-mediated exon skipping efficiency

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4123610A (en) * 1977-03-09 1978-10-31 The United States Government Nucleic acid crosslinking agent and affinity inactivation of nucleic acids therewith
US4507433A (en) * 1983-10-07 1985-03-26 The Johns Hopkins University Preparation of oligodeoxyribonucleoside alkyl or arylphosphonates

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US468950A (en) * 1892-02-16 Commutator-brush for electric motors and dynamos
US3211732A (en) * 1965-10-12 Pyrazolob:x-d]pyrlil/hdines
US3225042A (en) * 1962-07-31 1965-12-21 Lilly Co Eli Hydroxymorpholines
US3797414A (en) * 1970-10-29 1974-03-19 M Ahrend Canted vortex open pit incinerator
US4430496A (en) * 1980-01-17 1984-02-07 Varian Associates, Inc. Strong anion exchange composition and methods
AU543007B2 (en) * 1980-04-15 1985-03-28 Technicon Instruments Corportion Agglutination immunoassay
US4395486A (en) * 1981-08-19 1983-07-26 Medical College Of Ga. Research Inst., Inc. Method for the direct analysis of sickle cell anemia
US4515781A (en) * 1983-02-23 1985-05-07 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services 2',5'-Riboadenylate-morpholinoadenylate nucleotides
CA1254525A (en) * 1983-04-13 1989-05-23 Christine L. Brakel Kit for terminally chemically labeling dna
GB8311228D0 (en) * 1983-04-25 1983-06-02 Fujisawa Pharmaceutical Co Morpholine derivatives
US4713326A (en) * 1983-07-05 1987-12-15 Molecular Diagnostics, Inc. Coupling of nucleic acids to solid support by photochemical methods
CA1256005A (en) * 1983-09-26 1989-06-20 W. Peter Hansen Sandwich hybridization method for nucleic acid detection
US4734363A (en) * 1984-11-27 1988-03-29 Molecular Diagnostics, Inc. Large scale production of DNA probes
DE3515529A1 (en) * 1985-04-30 1986-11-06 Dr. Helmut Bayer Chemisch-technische Betriebsberatung, 6052 Mühlheim Process for the solid-phase synthesis of oligonucleotides of defined sequence
JPS62172384A (en) * 1986-01-24 1987-07-29 Sanyo Electric Co Ltd Toner concentration detecting device
JPH09118898A (en) * 1995-08-23 1997-05-06 Kao Corp Bleaching agent composition for herd surface

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4123610A (en) * 1977-03-09 1978-10-31 The United States Government Nucleic acid crosslinking agent and affinity inactivation of nucleic acids therewith
US4507433A (en) * 1983-10-07 1985-03-26 The Johns Hopkins University Preparation of oligodeoxyribonucleoside alkyl or arylphosphonates

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Biochemistry, Volume 18, No. 23, issued November 13, 1979, P.S. MILLER et al, "Nonionic Nucleic Acid Analogues. Synthesis and Characterization of Dideoxyribonucleoside Methylphosphonates", see page 5140 *
Biochemistry, Volume 20, No. 7, issued March 31, 1981, P.S. MILLER et al, "Biochemical and Biological Effects of Nonionic Nucleic Acid Methylphosphonates", see pages 1878 and 1879. *
Biochemistry, Volume 24, No. 22 issued August, 1985, A. MURAKAMI et al, "Characterization of Sequence-Specific Oligodeoxyribonucleoside Methylphosphonates and Their Interaction with Rabbit Globin mRNA", see pages 4045 and 4046. *
CHEMICAL ABSTRACTS, Volume 91, No. 19, issued 1979 (Columbus, Oregon, USA), J. SUMMERTON, "Intracellular Inactivation of Specific Nucleotide Sequences: a General Approach to the Treatment of Viral Diseases and Virally Mediated Cancers," see pages 73, column 2 and 74, column 1, the Abstract No. 151527g, J. Theor. Biol. 1979, 78 (1), 77-99 (Eng). *
Journal of Biological Chemistry, Volume 255, No. 20, issued October 25, 1980, P.S. MILLER et al, "Oligothymidylate Analogues Having Stereoregular, Alternating Methylphosphonate/Phosphodiester Backbones", see page 9659. *
Nucleic Acids Research, Volume 12, No. 8, issued April 25, 1984, M. RENZ et al, "A Colorimetric Method for DNA Hybridization", see page 3435. *

Cited By (156)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5185444A (en) * 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5470974A (en) * 1985-03-15 1995-11-28 Neu-Gene Development Group Uncharged polynucleotide-binding polymers
US5235033A (en) * 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
EP0281390A3 (en) * 1987-03-02 1990-04-25 J. Arnold Lyle Jr. Polycationic supports for nucleic acid purification, separation and hybridization
EP0281390A2 (en) * 1987-03-02 1988-09-07 Gen-Probe Incorporated Polycationic supports for nucleic acid purification, separation and hybridization
US4912199A (en) * 1987-07-06 1990-03-27 The Governors Of The University Of Alberta Oligopeptide anticancer and antiviral agents
US5126439A (en) * 1987-09-22 1992-06-30 Temple University Artificial dna base pair analogues
FR2627590A1 (en) * 1988-02-24 1989-08-25 Ire Celltarg Sa NUCLEIC ACID PROBE COMPRISING A CHEMICALLY MODIFIED 5 (PREMIUM) (OH) TERMINAL NUCLEOTIDE FOR NON-RADIOACTIVE MARKING AND METHODS OF PREPARATION
EP0334694A1 (en) * 1988-02-24 1989-09-27 La Region Wallonne Nucleic acid probes with non-radioactive labels, and preparation processes
US5218102A (en) * 1988-02-24 1993-06-08 Improbio Nucleic acid probe containing a terminal carbamyl linking non-radioactive labeling and preparating processes
EP0411186A1 (en) * 1989-08-01 1991-02-06 Abbott Laboratories DNA probe assay using neutrally charged probe strands
US6476205B1 (en) 1989-10-24 2002-11-05 Isis Pharmaceuticals, Inc. 2′ Modified oligonucleotides
US5792847A (en) * 1989-10-24 1998-08-11 Gilead Sciences, Inc. 2' Modified Oligonucleotides
US6911540B2 (en) 1989-10-24 2005-06-28 Isis Pharmaceuticals, Inc. 2′ Modified oligonucleotides
EP0962463A1 (en) * 1989-12-20 1999-12-08 Antivirals Inc. Uncharged morpholino-based polymers having phosphorus-containing chiral intersubunit linkages
AU655164B2 (en) * 1989-12-20 1994-12-08 Antivirals Inc. Uncharged morpholino-based polymers having phosphorous-containing chiral intersubunit linkages
EP0506845A4 (en) * 1989-12-20 1992-10-21 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
EP0506830B1 (en) * 1989-12-20 2005-12-14 Avi Biopharma, Inc. Uncharged morpholino-based polymers having phosphorous-containing chiral intersubunit linkages
EP0506845A1 (en) * 1989-12-20 1992-10-07 Antivirals Inc. Uncharged morpholino-based polymers having achiral intersubunit linkages
EP0506830A1 (en) * 1989-12-20 1992-10-07 Antivirals Inc. Uncharged morpholino-based polymers having phosphorous-containing chiral intersubunit linkages
US5378841A (en) * 1989-12-20 1995-01-03 Antivirals Inc. Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5969118A (en) * 1990-07-27 1999-10-19 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and preparation thereof through radical coupling
US6121433A (en) * 1990-07-27 2000-09-19 Isis Pharmaceuticals, Inc. Oligomeric compounds having nitrogen-containing linkages
US5783682A (en) * 1990-07-27 1998-07-21 Isis Pharmaceuticals, Inc. Oligonucleotide mimics having nitrogen-containing linkages
US6900301B2 (en) 1990-07-27 2005-05-31 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5965721A (en) * 1990-07-27 1999-10-12 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US6271357B1 (en) 1990-07-27 2001-08-07 Isis Pharmaceuticals, Inc. Oligonucleotide mimics having nitrogen-containing linkages
US5618704A (en) * 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5614617A (en) * 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5610289A (en) * 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
EP0549686A1 (en) * 1990-09-20 1993-07-07 Gilead Sciences, Inc. Modified internucleoside linkages
EP0549686A4 (en) * 1990-09-20 1995-01-18 Gilead Sciences Inc Modified internucleoside linkages
EP0565646A1 (en) * 1991-04-19 1993-10-20 Baylor College Of Medicine Nucleosides and oligonucleosides with a phosphate-free internucleoside backbone and process for preparing the same
EP0565646A4 (en) * 1991-04-19 1994-08-03 Baylor College Of Medicine
US5977296A (en) * 1991-05-24 1999-11-02 Nielsen; Peter Chiral peptide nucleic acid monomers and oligomers
EP1162206A2 (en) * 1991-05-24 2001-12-12 BUCHARDT, Ole Peptide nucleic acids
US7378485B2 (en) 1991-05-24 2008-05-27 Ole Buchardt Peptide nucleic acids with polyamide-containing backbones
US6613873B1 (en) 1991-05-24 2003-09-02 Isis Pharmaceuticals, Inc. Peptide nucleic acids having 2,6-diaminopurine nucleobases
US7223833B1 (en) * 1991-05-24 2007-05-29 Isis Pharmaceuticals, Inc. Peptide nucleic acid conjugates
WO1992020702A1 (en) * 1991-05-24 1992-11-26 Ole Buchardt Peptide nucleic acids
EP1162206A3 (en) * 1991-05-24 2004-04-14 BUCHARDT, Ole Peptide nucleic acids
EP1074559A1 (en) * 1991-05-24 2001-02-07 BUCHARDT, Ole Peptide nucleic acids
US6451968B1 (en) 1991-05-24 2002-09-17 Isis Pharmaceuticals, Inc. Peptide nucleic acids
US6201103B1 (en) 1991-05-24 2001-03-13 Peter E. Nielsen Peptide nucleic acid incorporating a chiral backbone
US6441130B1 (en) 1991-05-24 2002-08-27 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
WO1992020703A1 (en) * 1991-05-24 1992-11-26 Ole Buchardt The use of nucleic acid analogues in diagnostics and analytical procedures
US6710163B1 (en) 1991-05-24 2004-03-23 Ole Buchardt Peptide nucleic acid synthons
US6414112B1 (en) 1991-05-24 2002-07-02 Ole Buchardt Peptide nucleic acids having 2,6-diaminopurine nucleobases
US6357163B1 (en) 1991-05-24 2002-03-19 Ole Buchardt Use of nucleic acid analogues in diagnostics and analytical procedures
US5714331A (en) * 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5786461A (en) * 1991-05-24 1998-07-28 Buchardt; Ole Peptide nucleic acids having amino acid side chains
US5736336A (en) * 1991-05-24 1998-04-07 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
EP1411063A1 (en) * 1991-05-24 2004-04-21 BUCHARDT, Ole Peptide nucleic acids
US6713602B1 (en) 1991-05-24 2004-03-30 Ole Buchardt Synthetic procedures for peptide nucleic acids
US5766855A (en) * 1991-05-24 1998-06-16 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity and sequence specificity
US5773571A (en) * 1991-05-24 1998-06-30 Nielsen; Peter E. Peptide nucleic acids
US6395474B1 (en) 1991-05-24 2002-05-28 Ole Buchardt Peptide nucleic acids
US5877160A (en) * 1991-05-31 1999-03-02 Genta Incorporated Compositions and methods of treatment of androgen-associated baldness using antisense oligomers
WO1993000352A1 (en) * 1991-06-20 1993-01-07 Antivirals Inc. Sequence-specific binding polymers for duplex nucleic acids
AU665560B2 (en) * 1991-06-20 1996-01-11 Antivirals Inc. Sequence-specific binding polymers for duplex nucleic acids
US5792608A (en) * 1991-12-12 1998-08-11 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
US5359044A (en) * 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US6001841A (en) * 1991-12-13 1999-12-14 Isis Pharmaceuticals, Inc. Cyclobutyl oligonucleotide surrogates
EP0618923A1 (en) * 1991-12-18 1994-10-12 Glaxo Wellcome Inc. Peptide nucleic acids and their effect on genetic material
EP0618923A4 (en) * 1991-12-18 1998-05-20 Glaxo Inc Peptide nucleic acids and their effect on genetic material.
EP1044987A2 (en) * 1991-12-24 2000-10-18 Isis Pharmaceuticals, Inc. Gapped 2'-modified oligonucleotides
EP0618925A1 (en) * 1991-12-24 1994-10-12 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US5700922A (en) * 1991-12-24 1997-12-23 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
EP1044987A3 (en) * 1991-12-24 2001-10-04 Isis Pharmaceuticals, Inc. Gapped 2'-modified oligonucleotides
US6277603B1 (en) 1991-12-24 2001-08-21 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
EP0618925A4 (en) * 1991-12-24 1997-01-02 Isis Pharmaceuticals Inc Gapped 2' modified oligonucleotides.
WO1993022326A1 (en) * 1992-04-24 1993-11-11 Beckman Instruments, Inc. Protecting groups useful in oligonucleotide synthesis
US6228982B1 (en) * 1992-05-22 2001-05-08 Benget Norden Double-stranded peptide nucleic acids
US6410702B1 (en) 1992-06-01 2002-06-25 Isis Pharmaceuticals, Inc. Modified internucleoside linkages (II)
US5434257A (en) * 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US6683166B2 (en) 1992-06-01 2004-01-27 Isis Pharmaceuticals, Inc. Modified internucleoside linkages (ii)
US5817781A (en) * 1992-06-01 1998-10-06 Gilead Sciences, Inc. Modified internucleoside linkages (II)
EP1258492A1 (en) * 1992-09-24 2002-11-20 Chiron Corporation N-substituted oligomers and methods for their synthesis, e.g. polyglycine bearing nucleic acid bases
WO1994012517A1 (en) * 1992-11-23 1994-06-09 Antivirals Inc. Sequence-specific binding polymers for duplex nucleic acids
WO1994022891A1 (en) * 1993-03-31 1994-10-13 Sterling Winthrop Inc. Oligonucleotides with amide linkages replacing phosphodiester linkages
EP1310507A3 (en) * 1993-04-26 2004-03-17 NIELSEN, Peter Eigil Novel peptide nucleic acids
WO1994025477A2 (en) * 1993-04-26 1994-11-10 Nielsen Peter E Novel peptide nucleic acids
WO1994025477A3 (en) * 1993-04-26 1994-12-22 Peter E Nielsen Novel peptide nucleic acids
US7825215B1 (en) * 1993-04-26 2010-11-02 Peter E. Nielsen Substituted nucleic acid mimics
EP1310507A2 (en) * 1993-04-26 2003-05-14 NIELSEN, Peter Eigil Novel peptide nucleic acids
WO1995001369A1 (en) * 1993-07-02 1995-01-12 Ole Buchardt Double-stranded peptide nucleic acids
US5623049A (en) * 1993-09-13 1997-04-22 Bayer Aktiengesellschaft Nucleic acid-binding oligomers possessing N-branching for therapy and diagnostics
US7375198B2 (en) 1993-10-26 2008-05-20 Affymetrix, Inc. Modified nucleic acid probes
US6156501A (en) * 1993-10-26 2000-12-05 Affymetrix, Inc. Arrays of modified nucleic acid probes and methods of use
US7794943B2 (en) 1993-10-26 2010-09-14 Affymetrix, Inc. Modified nucleic acid probes
US6710164B1 (en) 1993-11-22 2004-03-23 Peter E. Nielsen Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5719262A (en) * 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US6133444A (en) * 1993-12-22 2000-10-17 Perseptive Biosystems, Inc. Synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions
US6172226B1 (en) 1993-12-22 2001-01-09 Perseptive Biosystems, Inc. Synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions
EP0746326A1 (en) * 1994-01-11 1996-12-11 Isis Pharmaceuticals, Inc. Oligomeric compounds having nitrogen-containing linkages
EP0746326A4 (en) * 1994-01-11 2000-06-14 Isis Pharmaceuticals Inc Oligomeric compounds having nitrogen-containing linkages
WO1995031459A1 (en) * 1994-05-11 1995-11-23 Antivirals Inc. Morpholino-subunit combinatorial library and method
WO1996002558A1 (en) * 1994-07-15 1996-02-01 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
US5705333A (en) * 1994-08-05 1998-01-06 The Regents Of The University Of California Peptide-based nucleic acid mimics(PENAMS)
US5955571A (en) * 1994-08-08 1999-09-21 Bayer Aktiengesellschaft Nucleic acid-binding oligomers for therapy and diagnosis
WO1996005215A1 (en) * 1994-08-17 1996-02-22 The Perkin Elmer Corporation Solid support reagents for the synthesis of 3'-nitrogen containing polynucleotides
US6045995A (en) * 1994-08-24 2000-04-04 Isis Pharmaceuticals, Inc. Capillary electrophoretic detection of nucleic acids
WO1996006189A1 (en) * 1994-08-24 1996-02-29 Isis Pharmaceuticals, Inc. Capillary electrophoretic detection of nucleic acids
US5874213A (en) * 1994-08-24 1999-02-23 Isis Pharmacueticals, Inc. Capillary electrophoretic detection of nucleic acids
WO1996014330A1 (en) * 1994-11-02 1996-05-17 Icnpharmaceuticals Amino acid nucleic acids
US6559303B1 (en) 1995-01-11 2003-05-06 Isis Pharmaceuticals, Inc. Methods for processing chemical compounds having reactive functional groups
US6670393B2 (en) 1995-06-07 2003-12-30 Promega Biosciences, Inc. Carbamate-based cationic lipids
US6063569A (en) * 1995-06-07 2000-05-16 Perseptive Biosystems, Inc. Methods for automated synthesis of PNA-DNA chimeras and compositions thereof
US6514693B1 (en) 1995-10-12 2003-02-04 Tetramerics Biotechnology, Inc. Method for detecting multiple copies of a repeat sequence in a nucleic acid molecule
EP0885238A1 (en) * 1996-03-08 1998-12-23 NIELSEN, Peter Eigil Substituted nucleic acid mimics
US7569659B1 (en) 1996-03-08 2009-08-04 Peter E. Nielsen Admixture comprising a target molecule and a nucleic acid mimic comprising sterically bulky substituents
EP0885238A4 (en) * 1996-03-08 2005-02-09 Nielsen Peter Eigil Substituted nucleic acid mimics
WO1997032888A1 (en) * 1996-03-08 1997-09-12 Isis Pharmaceuticals, Inc. Substituted nucleic acid mimics
US7695902B2 (en) 1996-06-06 2010-04-13 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US7812149B2 (en) 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US7919612B2 (en) 1996-06-06 2011-04-05 Isis Pharmaceuticals, Inc. 2′-substituted oligomeric compounds and compositions for use in gene modulations
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
WO1998003542A1 (en) * 1996-07-24 1998-01-29 Buchardt, Dorte Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5908845A (en) * 1996-10-30 1999-06-01 Segev; David Polyether nucleic acids
EP0839830A1 (en) * 1996-10-30 1998-05-06 Bio-Rad Laboratories, Inc. Polyether nucleic acids
US6476215B1 (en) * 1997-08-01 2002-11-05 Canon Kabushiki Kaisha Ink jet method of spotting a probe and manufacturing a probe array
US7994297B2 (en) 1997-08-01 2011-08-09 Canon Kabushiki Kaisha Method of spotting probes on a solid support
WO1999011820A1 (en) * 1997-09-02 1999-03-11 Isis Pharmaceuticals, Inc. Compositions and methods for the identification and quantitation of deletion sequence oligonucleotides in synthetic oligonucleotide preparations
US6734161B1 (en) 1997-09-16 2004-05-11 Peter E. Nielsen Peptide nucleic acids having antibacterial activity
US6300318B1 (en) 1997-09-16 2001-10-09 Peter E. Nielsen Antibacterial and antibiotic methods using peptide nucleic acids and pharmaceutical compositions therefor
WO1999013893A1 (en) * 1997-09-16 1999-03-25 Isis Pharmaceuticals, Inc. Peptide nucleic acids having antibacterial activity
US6433134B1 (en) 1998-07-09 2002-08-13 Biocept, Inc. Peptide nucleic acid precursors and methods of preparing same
WO2000044897A1 (en) * 1999-01-29 2000-08-03 Avi Biopharma, Inc. Method of treating restenosis by antisense targeting of c-myc
WO2001016365A1 (en) * 1999-08-30 2001-03-08 Bio-Rad Laboratories, Inc. Poly(ether-thioether), poly(ether-sulfoxide) and poly(ether-sulfone) nucleic acids
JP2003508062A (en) * 1999-08-30 2003-03-04 バイオ−ラッド ラボラトリーズ，インコーポレイティド Poly (ether-thioether) nucleic acids, poly (ether-sulfoxide) nucleic acids and poly (ether-sulfone) nucleic acids
US6348583B1 (en) * 1999-08-30 2002-02-19 Bio-Rad Laboratories, Inc. Poly(ether-thioether), poly(ether-sulfoxide) and poly(ether-sulfone) nucleic acids
US7348148B2 (en) 2001-01-29 2008-03-25 Bio-Rad Laboratories Inc. Nucleic acid derivatives
US7683164B2 (en) 2001-01-29 2010-03-23 Bio-Rad Laboratories Inc. Nucleic acid derivatives
US7034131B2 (en) 2001-01-29 2006-04-25 Bio-Rad Laboratories Inc. Nucleic acid derivatives
US7241890B2 (en) 2001-10-30 2007-07-10 Conforma Therapeutics Corporation Purine analogs having HSP90-inhibiting activity
US9150605B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
WO2004044137A2 (en) * 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Modified oligomeric compounds and compositions for use in gene modulation
US9150606B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
WO2004044137A3 (en) * 2002-11-05 2005-05-12 Isis Pharmaceuticals Inc Modified oligomeric compounds and compositions for use in gene modulation
US8124745B2 (en) 2002-11-05 2012-02-28 Isis Pharmaceuticals, Inc Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US8604183B2 (en) 2002-11-05 2013-12-10 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US7696345B2 (en) 2002-11-05 2010-04-13 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US7262192B2 (en) 2003-04-29 2007-08-28 Pfizer Inc. Substituted pyrazolo[4,3-d]pyrimidines and their use as PDE-5 inhibitors
US7129244B2 (en) 2003-09-18 2006-10-31 Conforma Therapeutics Corporation Triazolopyrimidines and related analogs as HSP90-inhibitors
US7148228B2 (en) 2003-09-18 2006-12-12 Conforma Therapeutics Corporation Pyrazolopyrimidines and related analogs as HSP90-inhibitors
US7138402B2 (en) 2003-09-18 2006-11-21 Conforma Therapeutics Corporation Pyrrolopyrimidines and related analogs as HSP90-inhibitors
US7138401B2 (en) 2003-09-18 2006-11-21 Conforma Therapeutics Corporation 2-aminopurine analogs having HSP90-inhibiting activity
US7125945B2 (en) 2003-09-19 2006-10-24 Varian, Inc. Functionalized polymer for oligonucleotide purification
US7572799B2 (en) 2003-11-24 2009-08-11 Pfizer Inc Pyrazolo[4,3-d]pyrimidines as Phosphodiesterase Inhibitors
US8097621B2 (en) 2003-11-24 2012-01-17 Pfizer Inc. Pyrazolo[4,3-d]pyrimidines as phosphodiesterase inhibitors
US8569474B2 (en) 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
US7569572B2 (en) 2004-04-07 2009-08-04 Pfizer Inc Pyrazolo[4,3-D]pyrimidines
US8394947B2 (en) 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
US8093229B2 (en) 2005-03-30 2012-01-10 Conforma Therapeutics Corporation Alkynyl pyrrolo[2,3-d]pyrimidines and related analogs as HSP90-inhibitors
US7544672B2 (en) 2005-03-30 2009-06-09 Conforma Therapeutics Corporation Alkynyl pyrrolo[2,3-d]pyrimidines and related analogs as HSP90-inhibitors

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DE3650349D1 (en) 1995-08-17
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ATE81872T1 (en) 1992-11-15
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EP0639582B1 (en) 1998-09-16
DE3687030T2 (en) 1993-03-11
JP3022967B2 (en) 2000-03-21
WO1986005519A1 (en) 1986-09-25
ATE124999T1 (en) 1995-07-15
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EP0215942A4 (en) 1988-08-23
DE3650349T2 (en) 1995-12-14
EP0216860A4 (en) 1989-02-22
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JP2670434B2 (en) 1997-10-29
EP0215942A1 (en) 1987-04-01
DE3687030D1 (en) 1992-12-03
DE3650699T2 (en) 1999-04-15
CA1268404A (en) 1990-05-01
EP0216860A1 (en) 1987-04-08
JPH08193998A (en) 1996-07-30
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EP0216860B1 (en) 1992-10-28
JP2528107B2 (en) 1996-08-28

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